MX2008010285A

MX2008010285A - Gene expression system using alternative splicing in insects.

Info

Publication number: MX2008010285A
Application number: MX2008010285A
Authority: MX
Inventors: Luke Alphey
Original assignee: Oxitec Ltd
Priority date: 2006-02-10
Filing date: 2007-02-12
Publication date: 2009-01-16
Also published as: CN101421408A; AU2007213444A1; AU2007213444B2; EP1984512B1; EP3159414A1; US20090183269A1; WO2007091099A1; EP1984512A1

Abstract

A polynucleotide expression system is provided that is capable of alternative splicing of RNA transcripts of a polynucleotide sequence to be expressed in an organism.

Description

SYSTEM OF GENETIC EXPRESSION USING AN ALTERNATIVE SPLICE IN INSECTS Field of the Invention The present invention relates to a gene expression system, in combination with splicing control sequences, the control sequences provide a mechanism for alternative splicing. Background of the Invention All references cited herein are incorporated by reference, unless otherwise evident. Alternative splicing includes the removal of one or more introns and the linking of the flanking exons. This reaction is catalyzed by the spliceosome, a macromolecular machine composed of five RNAs and hundreds of proteins (Jurica, M. S. &Moore, M. J. (2003) Mol.Cell 12, 5-14). Alternative splicing generates multiple mRNAs from a single gene, thus increasing the diversity of proteomes (Graveley, B. R. (2001) Trends Gent., 17, 100-107). Alternative splicing also plays an important role in the regulation of gene expression in many developmental processes ranging from sex determination to apoptosis (Black, DL (2003) Annu Rev. Biochem. 72, 291-336) and defects in alternative splicing have been associated with many human problems (Caceres, JF &Kornblihtt, AR (2002) Trends Gent., 18, 186-193). In general, the alternative splice is it regulates by means of the proteins that are associated with the pre-mRNA and works to better or repress the ability of the spiiceosome to recognize the splice sites that flank the regulated exon (Smith, CW &Valcarcel, J. (2000) Trends Biochem Sd. 25, 381-388). When a particular alternative exon will be included or excluded from the mature RNA in each cell it is believed that it is determined by means of the relative concentration of a number of positive and negative splicing regulators and the interactions of those factors with the pre-mRNA and the Spiiceosome components (Smith, CW &Valcarcel, J. (2000) Trends Biochem, Sd. 25, 381-388). Spliceosomes are large complexes of small nuclear RNAs and protein particles (SnRNP) that assemble with pre-mRNA, to produce RNA splicing, by removing introns from eukaryotic nuclear RNAs, producing mRNA that then moves to protein in the ribosomes. Although at least 74% of the human genes encode alternatively spliced mRNAs (Smith, C.W. &Valcarcel, J. (2000) Trends Biochem.Sd. 25, 381-388), relatively few splicing regulators have been identified. Detailed Description of the Invention Thus, in a first aspect, the present invention provides a polynucleotide expression system comprising: at least one heterologous polynucleotide sequence encoding a functional protein, defined between a start codon and a stop codon, and / or polynucleotides for interfering RNA (RNAi), which is to be expressed in an organism; at least one promoter operatively linked; and at least one splice control sequence that in cooperation with a spliceosome, is capable of (i) mediating the splicing of an RNA transcript of the coding sequence to yield a first spliced messenger RNA product (mRNA) and (ii) mediating at least one alternative splicing of the RNA transcript to produce an alternative spliced mRNA product; wherein when at least one heterologous polynucleotide sequence encodes a functional protein, at least one of the mature mRNA products comprises an open reading frame (ORF) that extends from the start codon to the stop codon, defining a protein which is the functional protein, or is related to the functional protein by means of at least one amino acid deletion and which is functional when transferred and optionally has undergone a post-translational modification; the measurement is selected from the group consisting of: sex-specific mediation, stage-specific mediation, mediation specific to the germ line, mediation specific to the tissue, and their combinations. The expression system can be DNA or RNA or a hybrid or combination of both. It is envisaged that the system comprises both ribo- and deoxy-ribonucleotides, that is, portions of DNA and portions of RNA. These could correspond to different genetic elements, in the way that the system is a DNA / RNA hybrid, with some functional elements provided by the DNA and the others by the RNA. Preferably, the mediation is carried out in a specific manner to the sex, specific to the stage, specific to the germinal line or specific to the tissue. In particular, sex-specific measurement is preferred. However, it is preferred that a combination of these four modes of mediation can be used. It is particularly preferred that when a combination of those modes is used, that this includes sex-specific mediation. A particularly preferred example of such a combination is a combination of mediation specific to sex, tissue and the alternative splicing stage. The system can be adapted for the expression of a gene.

Preferably, the polynucleotide sequence to be expressed comprises a coding sequence for a protein or a polypeptide, that is, at least one exon and preferably two or more exons, capable of encoding a polypeptide, such as a protein or a fragment thereof. It will be understood that an exon is any region of DNA within a gene that is present in a mature molecule of RNA derived from that gene, and has not been unpaired from the transcribed RNA molecule. For the genes encoding the protein, the mature RNA molecules correspond to the mRNA molecules mature, which can encode one or more proteins or polypeptides. The exons can be eukaryotic genes interpolated with non-coding DNA segments. The at least one heterologous polynucleotide sequence can encode a functional protein, defined between a start codon and a stop codon to be expressed in an organism. Those sequences that are going to be expressed in the organism can also be called sequences, whose expression must be regulated in the organism. Preferably, the polynucleotide sequence to be expressed comprises two or more coding exons, which are segments or sequences of polynucleotides that encode amino acids when translated from the mRNA. Preferably, the different exons are differentially spliced together to provide alternative mRNAs. Preferably those alternative spliced mRNAs have different coding potential, that is they encode different proteins or polypeptide sequences. Thus the expression of the coding sequence is regulated to the splice alternatively the aforementioned ways of mediation. The polynucleotide sequence to be expressed may comprise polynucleotides for interfering RNA (RNAi). These sequences are capable of providing, for example, one or more extensions of double-stranded RNA (dsRNA), preferably in the form of a primary transcript, which in turn is capable of being processed by means of the "shredder" of RNA-like enzyme Pol.

III. These extensions include for example single-stranded RNA extensions that can form circuits such as those found in short-edge RNAs (shRNA), or with longer regions that are substantially self-complementary. Thus when the system is DNA, the polynucleotides for the interfering RNA are deoxyribonucleotides that when transcribed in pre-RNA reibonucleotides, provides an extension of dsRNA, as described above. Polynucleotides for interfering RNA are particularly preferred when the polynucleotides are positioned to minimize with alternative splicing. This can be achieved by means of the distal positioning of these polynucleotides from alternative splice control sequences, preferably 3 'to the control sequences. In another preferred embodiment, the substantially self-complementary regions can be separated from one another by means of one or more splice control sequences, such as an intron, mediating the alternative splice. Preferably, the self-complementary regions are placed as a series of two or more inverted repeats, each inverted repeat is separated by the splicing control sequence, preferably an intron, as defined above. In this configuration, the different alternately spliced transcripts may have their substantially self-complementary regions separated by different non-self-complementary sequence len in the mature transcript (post-splicing) alternative). It will be appreciated that regions that are substantially self-complementary are those that are capable of forming orbits for example as portions of the sequence that are capable of forming base pairs with other portions of the sequence. Those two portions do not have to be exactly complementary to each other, since there may be mismatches or tolerance of extensions in each portion that do not form base pairs with each other. These extensions may not have an equivalent in another portion such that symmetry is lost and "bubbles" are formed, as is generally known with the complementation of base pairs. In another preferred embodiment, one or more segments of the sequence substantially complementary to another section of the primary transcript is placed, relative to the at least one splicing control sequence, such that it is not included in all transcripts produced at splice alternately of the primary transcript. By means of this method, some transcripts are produced that tend to produce dsRNA while others do not; by means of alternative splicing medication, this is sex-specific mediation, step-specific mediation, mediation specific to the germ line, tissue-specific mediation, and combinations thereof. The system is preferably capable of expressing at least one protein of interest, that is, the functional protein to be expressed in an organism. That's when you pair a protein interest may have a therapeutic effect or may, preferably, be a scavenger, for example DsRed, green fluorescent protein (GF) or one or more of its mutants or variants, or other markers that are well known in the art. More preferably, the functional protein to be expressed in an organism has a lethal, damaging or sterilizing effect. When reference is made to a lethal effect, it will be appreciated that this extends to a damaging or sterilizing effect, such that it is capable of killing an organism per se or its offspring, or capable of reducing or destroying the function of certain tissues, of the which reproductive tissues are particularly preferred, in such a way that the organism or its offspring is sterile. Therefore some lethal effects such as poisons kill the organism or tissue in a short period of time in relation to their life time, while others simply reduce the ability of the organism to function, for example to reproduce. A resulting lethal effect on sterilization is particularly preferred since this allows the organism to complete in the wild ("in nature") with native type organisms, but the sterile insect can not produce viable offspring. In this way the present invention achieves a result similar to techniques such as the sterile insect technique (SIT) in insects, without the problems associated with SIT, such as cost, danger to the user, and reduced organism competitiveness. radiated.

Preferably, the system comprises at least one positive feedback mechanism, in fact at least one functional protein to be differentially expressed by alternative splicing and at least one promoter thereof, wherein a product of a gene to be expressed serves as a positive transcription control factor for at least one promoter, and where the product or expression of the product is controllable. Preferably, a reinforcer is associated with the promoter, the genetic product serves to improve the activity of the promoter by means of the enhancer. Preferably the control factor is the tTA gene product or its analog, and wherein one or more tetO operator units is operably linked to the promoter and its enhancer, tTA or its analog serve to enhance the activity of the promoter via tetO. It is preferred that the functional protein encode the product TAV or tTAF and preferably the promoter is substantially inactive in the absence of the positive transcription control factor. It is appropriate that the promoters for this system, preferably minimums are selected from: hsp70, a minimal promoter P, a minimal CMV promoter, a minimal promoter based on SAct5C, a BmA3 promoter fragment, a humpback promoter fragment, an Adh core promoter, and a minimal promoter Act4C, or its combinations. In one embodiment, the functional protein is preferably an apoptosis-inducing factor such as the AIF protein described for example by Cande et al. (Journal of Cell Science 115, 4727-4734 (2002)) or their counterparts. AIF homologs are found in mammals and even in invertebrates including insects, nematodes, fungi, and plants, which means that the AIF gene has been conserved in the eukaryotic kingdom. Also preferred is Hid, the protein product of the defective gene of the superior involution of Drosophila melanogaster, or reaper (Rpr), the product of the Drosophila harvester gene or its mutants. The use of Hid was described by Heinrich and Scott (Proc. Nati Acad. Sci USA 97, 8229-8232 (2000) .The use of a mutant derivative, HidA'a5 was described by Horn and Wimmer (Nature Biotechnology 21, 64- 70 (2003)) Use of a mutant derivative of Rpr, RprKR, is described here (see also White and others 1996, Wing et al., 2001, and Olson et al., 2003). Both Rpr and Hid are proprotein proteins. -apópticas, which are believed to bind to IAP1, IAP1 is an anti-apoptic protein, Hid and Rpr are therefore expected to work across a broad phylogenetic range (Huang et al., 2002, Vernooy et al., 2000 ) even though its own sequence is not well conserved NippIDm, the mammalian Drosophila homolog Nippl (Parker and others Biochemical Journal 368, 789-797 (2002); Bennett et al., Genetics 164, 235-245 ( 2003).) NippIDm is another example of a protein with a lethal effect expressed at an adequate level as understood by the expert. protein lozenges with lethal effect will be well known to those skilled in the art. It is also preferred that the functional protein itself be a transcriptional transactivator such as the tTAV system described above. It is preferred that the promoter can be activated by environmental conditions, for example the presence or absence of a particular factor such as tetracycline in the tet system described herein, such that the expression of the gene of interest can be easily manipulated by someone skilled in the art. Alternatively, a preferred example of a suitable promoter is the hsp70 heat shock promoter, which allows the user to control the expression by varying the ambient temperature at which the hosts are exposed in a laboratory or in the field for example. . Another preferred example of temperature control is described by Fryxell and Miller (Journal of Economic Entomology 88, 1221-1232 (1995)). Also preferred as a promoter is the srya promoter specific to the embryo (Horn &Wirnmer (2003), of Drosophila melanogaster, or its homologs or promoters of other genes specific or active of embryos such as the Drosophila slow as molasses (slam) gene). It is also preferred that the system comprises other factors upstream 5 'and / or downstream 3' to control expression.Examples include reinforcers such as fat body boosters of the yolk protein genes. Drosophila, and baculovirus homology region (hr) reinforcers, for example / AcMNPV, will also appreciate that the products will include UTR 5 'and 3', for example. The splicing control sequence allows for an additional level of control of protein expression, in addition to the promoter and / or enhancer of the gene. For example, tissue-specific or sex-specific expression of insect embryos would be extremely difficult by conventional methods. Promoters with this specificity are unknown even in Drosophila. However, when using combination control according to the present invention, an embryo-specific promoter, for example srya, can be combined with a suitable alternative splicing system. It is preferred to consider any combination of promoter and alternative splicing mechanism. The promoter is preferably specific to a particular protein having a short temporal or confined spatial effect, for example a cellular autonomous effect. Alternatively it is preferred that the promoter be specific for a broader class of proteins or a specific protein having a long term and / or systemic broad effect, such as a hormone, positive or negative growth factor, morphogen and another secreted molecule or signaling of the cell surface. This would allow for example a higher expression pattern such that a combination of a morphogenic promoter with an alternative splicing mechanism specific to the step could result in the morphogen expressing only one Once a certain stage in the life cycle has been reached, but the effect of the morphogen would still be felt (this is the morphogen can still act and have an effect) beyond that stage of the life cycle. Preferred examples would be the morphogen / signal molecules of hedgehog, wigless / WNT,? T? B /?, EGF and their homologs, which are well-known signaling molecules in evolution. It is also envisaged that a promoter that is activated by a range of protein factors, for example transactivators or having a broad systemic effect, such as a hormone or morphogen, could be used in combination with an alternative splicing mechanism to obtain specific control to the tissue and to the eye or a specific control to the sex and to the stage, or a combination of specific control to the stage, the tissue, the germinal line and the sex. It is also envisaged that more than one promoter and optionally one enhancer may be used in the present system, either as alternative means for the initial transcription of the same protein or by virtue of the fact that the genetic system comprises more than one genetic expression system. (This is more of a gene and its promoter that accompanies it). In another aspect, the present invention provides a transformation method that you choose to express two or more RNA molecules, derived from a single primary transcript or substantially similar primary transcripts, by splicing alternative, those two or more RNA molecules that preferably encode different proteins or polypeptides, in an organism by contacting the organism with the expression system and preferably inducing the expression of the expression system. Methods of induction or transformation of the generic system and induction of expression are well known in the art with respect to the relevant organism. Transformed organisms (or transformants) are also provided through the present system. When reference is made to a nucleotide or a protein sequence, it will be understood that it includes reference to any mutant or variant thereof, which has a substantially equivalent biological activity. Preferably, the mutant or variant has at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 99%, preferably at least 99.9%, and more preferably at least 99.99% sequence identity with the sequences reference. The sequences provided can tolerate some sequence variation and still splice correctly. There are few nucleotides that are known to be important. These are the ones required for the entire splice, for example as shown in Figure 34. The initial GU and the final AG of the intron are particularly important and are therefore preferred, as described elsewhere, although approximately 5% of the introns start in GC, This consensus sequence is preferred although it is applied to all splices not specifically to alternative splices. In Figure 34, PU = A or G; PY = C or U. Preferably the system is or consists of a plasmid. As mentioned before, this can be a DNA, or RNA or a mixture of both. If the system comprises RNA, which may be preferred to reverse translate RNA into DNA by means of reverse transcriptase. If the reverse transcriptase is required, then the system may also comprise a coding sequence for the RT protein and a suitable promoter. Alternatively the RTase and the promoter can therefore be provided in a separate system such as a virus. In this case the system would only be activated after infection with that virus. The need to include cis-acting sequences suitable for reverse transcriptase or RNA-dependent RNA polymerase will be apparent to those skilled in the art. However, it is particularly preferred that the system be predominantly DNA and more preferably consist only of DNA, at least with respect to the sequences to be expressed in the organism. Although in other embodiments the at least one heterologous polynucleotide sequence to be expressed in an organism is a polynucleotide sequence capable of encoding a functional protein. The description will predominantly focus on the polynucleotide sequences that encode a functional protein but it will be understood that this also refers to polynucleotides for the interfering RNA (RNAi) unless the contrary is evident. It will be understood that reference is made to the start and stop codons between which the polynucleotide sequence to be expressed in an organism is defined, but which does not exclude placing at least one splicing control sequence, its elements or other sequences , such as introns in this region. In addition, the splicing control sequence, for example, may overlap with the start codon at least, in the sense that G of the ATG may be in some embodiments the initial 5'G of the splice control sequence. Thus the term "between" can be considered to refer to from the beginning (3 'to the initial nucleotide, that is A) of the initial codon, preferably 3' to the second nucleotide of the start codon (this is T) up to the first side nucleotide of the stop codon. Alternatively, as will be evident simply by reading a polynucleotide sequence, the stop codon may also be included. The at least one heterologous polynucleotide sequence to be expressed in an organism is a heterologous sequence. By "heterologous", it would be understood that it refers to a sequence that in the native type would not be found in association with, or linked to, at least one element or component of the at least one splicing control sequence. For example, where the splicing control sequence is derived from an organism particular and the heterologous polynucleotide is a coding sequence for a protein or a polypeptide, ie a polynucleotide sequence encoding a functional protein, then the coding sequence could be derived, in part or totally, from a gene of the same organism, with the condition that the origin of at least a part of the transcribed polynucleotide sequence was not the same as the origin of at least one splicing control sequence. Alternatively, the coding sequence could be from a different organism, and in this context, it could be thought to be "exogenous". The heterologous polynucleotide could also be considered as "recombinant", in that the sequence encoding a protein or polypeptide are derived from different places, either within the same genome (ie the genome of a single species or sub-species) or from different genomes (this is genomes of different species or subspecies). Heterologous may refer to a sequence other than the splicing control sequence, and therefore relate to the fact that the promoter and other sequences such as 5'UTR and / or 3'UTR may be heterologous to the polynucleotide sequence that is going to expressed in the organism, with the proviso that the polynucleotide sequence is not in association or operatively linked to the 5'UTR and / or 3'UTR promoter in the native type, ie the natural context of the polynucleotide sequence, if there is. It will be understood that heterolgo also applies to "designer" or hybrid sequences that do not derive from a particular organism but they are based on a number of components from different organisms and this would also satisfy the requirement that the sequence and at least one component of the splicing control sequence are not linked or found in association with the native type, even if a part or element of the hybrid sequence is thus, if at least one part or element is not. Preferably, a portion of at least 50 nucleotides of the hybrid sequence is not found in association with at least one component of the splicing control sequence, more preferably 200 nucleotides and most preferably 500 nucleotides. It will be understood that synthetic versions of natural sequences are also considered. Those synthetic sequences are also considered heterologous unless they are the sequence identical to a sequence that in the native type or in the natural context, would normally be in association with or associated with at least one element or component of at least one splicing control sequence. This also applies when the heterologous polynucleotide is a polynucleotide for the interfering RNA. In one embodiment, in which the polynucleotide to be expressed consists of a sequence to be expressed comprises a coding sequence for a protein or polypeptide, it will be understood that reference to expression in an organism refers to the provision of one or more transcribed RNA sequences, preferably mature mRNA, but this may preferably also refer to polypeptides translated into that organism. RT-PCR that demonstrates the presence of a transcript, not a protein, can be used to identify the transcribed RNA sequences. This is also particularly useful when the protein itself is not transferred or is not functional or is not identifiable by the antibodies raised against the native protein or native type, due to RNAi, post-translational modification or distorted folding. In another embodiment, in which the polynucleotide sequence to be expressed comprises polynucleotides for the interfering RNA, it will also be understood that the reference to the expression in an organism refers to the interaction of the polynucleotides for the interfering RNA, or transcripts. in the RNAi path, for example when linking the shredder or formation of small interfering ARs (siRNA). In fact it is particularly preferred that the polynucleotides for the interfering RNA comprise siRNA sequences and therefore have a length of 20-25 nucleotides, especially when the organism is a mammal. In insects or nematodes especially, it is preferred to provide portions of dsRNA, for example by means of the formation of orillas that can be processed by the shredder system. Mammalian cells generally produce an interferon response against the long dsRNA sequences, so that for mammalian cells it is more common to provide shorter sequences such as siRNA. Antisense sequences or sequences that have homology to the microRNAs that are naturally occurring RNA molecules that target the 3'UTR of protein are considered to be the sequences for RNAi according to one embodiment of the present invention. Each splice control sequence in the system comprises at least one splice acceptor site and at least one splice donor site. The number of donor and acceptor sites may vary depending on the number of segments of the sequence that are going to equalize each other. Preferably, the branching sites are included in each splicing control sequence. A branching site is the sequence to which the splice donor initially binds, see figure 32, which shows that splicing occurs in two stages, in which exon 5 'is separated and then attached to exon 3'. Referring to that figure the A is the only essential nucleotide and therefore is preferably included. Not wanting to limit the theory, it is believed that the pre-mRNA splice advances through the lartiato intermediate, as it does in the self-splicing of group II. First the break occurs at the 5 'junction, sometimes called the splice donor site. The phosphate at the 5 'end of the intron then binds to 2 ?? of an adenine approximately 25 nucleotides upstream of the 30th end of the intron, which is sometimes referred to as the acceptor site. This residue A is called the branch point. The next stage is that the break occurs in the union 3 'splice and the 5' phosphate of the downstream exon binds to the exon 3 'OH of the upstream exon. It is particularly preferred that the manner or mechanism of the alternative splice is sex specific. Preferably, the splicing control sequence is derived from an intron tra. However, it is particularly preferred that the alternative splicing mechanism is derived from the Cetra transforming gene from the Mediterranean fly, or from another ortholog or homologous of the Drosophila transforming gene, preferably C, pink or B. zonata especially one derived from the fruit fly tefritida. It is also preferred that the splicing control sequence is derived from the alternative splicing mechanism of the Actin-4 gene, in particular from Aedes spp and more preferably from AaActin-4, which is an Aedes / Stegomyia aegypti gene that shows specific splicing tissue, stage and sex. Preferably the alternative splice, particularly mediated by Actin-4k can add sequences that affect translation or stability of RNA, for example. It is also preferred that the splicing mechanism comprises a doublesex (dsx) gene fragment that is derived from Drosophila, B. mori, rose worm, apple moths, or a mosquito, in particular A. gambiae or especially A. aegypti . It is particularly preferred that the splicing control sequence is derived from dsx (preferably minigene 1 as written in the examples and is represented in the SEQ ID sequences.

DO NOT. 149 (the exons are present at positions 1-135, 1311-2446 and 3900-4389 of SEQ ID NO.149) which are included in the LA3491 construction) or Actin-4. Particularly preferred examples of the present invention are provided in the examples and can be selected from the group consisting of plasmids or constructs, in particular any according to any of Figures 19-31, especially any of the plasmids presented in Figures 16-18, 22-24, 26-32, 49, 52-55, and 61-69, and / or SEQ ID NOS. 46-48, 50-56, 143-145 and 151-162. Preferably, the functional protein to be expressed in an organism is tTAV, tTAV2 or tTAV3. Other proteins that are to be expressed in organisms are obviously considered in combination with the functional protein, preferably a lethal gene as described elsewhere. A continuous ORF can also be considered as an ORF, that is a polynucleotide sequence in mature mRNA, which does not include non-coding nucleotides, for example those that have the potential to be translated from amino acids. In this definition, it is preferred that the stop codon not be included. In some embodiments, the at least one splicing control sequence regulates the alternative splicing by intronic and exonic nucleotides. However, in one embodiment it is particularly preferred that the at least one control sequence of splicing is an intronic splicing control sequence. In other words, it is preferred that at least one splicing control sequence is derived substantially from the polynucleotides that are part of an intron and thus are cut from the primary transcript by splicing, such as those nucleotides that are not retained in the sequence of mature mRNA. Therefore, the intronic sequences can be considered as different from the "exonic" sequences that are retained in the RNA molecule processed (post-splicing). When the processed RNA molecule encodes a protein or an epitopeptide sequence, and is capable of being translocated, this has the correct structure and modifications such as a cap and a polyadenylation signal, for example it is known as mature or processed mRNA. and some of the exonic sequences then encode the amino acids when they move. It will be understood that in the alternative splice, the sequences can be intronic under some circumstances (this in other variants). Thus the at least one splicing control sequence of the present invention is preferably derived substantially from the polynucleotides that are part of an intron in at least one alternative splice variant, that is either in the first spliced mRNA product or the one when minus one alternatively spliced mRNA product. Thus introns or intronic sequences can be considered unpaired in at least one transcript or type of transcript.

For example consider the intron of C. capitata (intron Cetra, which is a particularly preferred example of at least one splicing control sequence according to the present invention.) According to Pane et al., Figure 2A, reproduced as Figure 33, all 7 of the Tra / Tra2 linkage sites highlighted are in the intronic sequence in the sense that they are found in portions of unblocked sequences in the F1 transcript, but on the other hand 6 out of 8 are exonic in the sense that they are found in exons that are included or retained in any M1 or M2 transcript, or both, so that Tra / Tra2 link types are intronic in this sense because they are able to control the alternative splice, but they are unblocked , this is not present, at least one alternate splice variant, this is at least one mRNA that has been spliced in an alternative way of the pre-RNA In the "normal" splicing (n or alternative) and in alternative splicing, the introns are generally removed from pre-RNA to form a spliced mRNA, which can then be translated into a polypeptide such as a protein or a protein fragment, having an amino acid sequence. Thus it will be readily apparent to those skilled in the art how to determine those sequences of the present system that would be considered intronic rather than exonic. Obviously that will appreciate that only a part of a mRNA actually moves, this is typically the part between the codon of start and the stop codon, although it will be understood that multiple starts and stoppages are sometimes present. Thus when reference is made to the translation of a mRNA sequence, it will be appreciated that it refers to the translation of the portion starting at the first nucleotide of the initial codon and ending after the last nucleotide before the start of the stop codon, which can considered as the coding portion. As mentioned, exonic sequences may be involved in the measurement of alternative splicing control, but it is preferred that at least some intron control sequences are involved in the mediation of the alternative splice. In other words, the genetic expression system of the present invention can also include splicing control sequences derived from or containing elements of the dsx gene, where, without wishing to be bound by theory, it is believed that the exonic sequences aid in the splicing mechanism alternative. Thus in some embodiments, the at least one splicing control sequence comprises the exonic sequence and it will be understood to be included in the definitions used to describe the present invention. As will be apparent, it is possible that some nucleotides are included within the definition of at least one splicing control sequence and also within the definition of a polynucleotide sequence that encodes a functional protein. In other words, the definition of these elements can overlap in such a way that certain nucleotides can be covered by the definition of more than one element. However, the person skilled in the art will recognize that this is not unusual in molecular biology since nucleotides frequently perform more than one paper. For example, in the present invention a nucleotide can be part of a coding sequence for a functional protein, but it could also be part of a recognized sequence and linked by a splicing factor, an example of which is the TRA protein or the TRA / TRA complex. , as described elsewhere. This is not unusual since for example some viruses have highly concentrated genomes where the same polynucleotide extension can encode two or up to three different proteins, each read in a different frame. Obviously, it may also be that the or the splicing control sequences are only intronic, that is without exonic influence. In fact this is particularly preferred. In some embodiments, it is preferred that at least one splicing control sequence be capable of being removed from the pre-RNA by splicing. Preferably, at least one splice control sequence does not result in a frame shift in at least one splice variant. Preferably it is a splice variant that encodes a full-length functional protein. In other words, the at least one splicing control sequence is not mediating the removal of the nucleotides that are part, or that are intended to be part of the sequence of polynucleotides encoding a functional protein, defined between a start codon and stop codon and / or the polynucleotides for interfering RNA (RNAi), to be expressed by an organism. By this is meant that the nucleotides are cut by splicing, in at least one splice variant, they are not nucleotides that encode the amino acids in the native type form of the protein or gene. One or more splice variants may have the nucleotides cut out, but at least one variant must retain those nucleotides, such that no frame shift is induced in the at least one variant. Those removed nucleotides are those that are removed in addition to sequences that are normally unblocked such as the intron. However, in view of the foregoing, it is also envisaged that different splice variants may result in the same sequence being read in different frames. Interaction by at least one splicing control sequence with cellular splicing machinery, for example the spliceosome, leads to or mediates the removal of a series of, preferably, at least 50 consecutive nucleotides from the primary transcript and the ligation (splicing) of the nucleotide sequences that were not consecutive in the primary transcript (because they, or their complement if the antisense sequence is considered, were not consecutive in the original sequence of the template from which the primary transcript was transcribed). The series of at least 50 consecutive nucleotides encompasses an intron. This mediation preferably acts in a sex-specific manner, specific to the stage, specific to the germinal line or specific to the tissue or its combinations, such that the equivalent primary transcripts in various sexes, stages, tissue types, etc., tend to to remove the introns of the different size or sequence, or can in some cases withdraw an intron in one case but not in others. This phenomenon, the removal of introns of different size or sequence in different circumstances, or the differentiated removal of introns of a given size or sequence, in different circumstances, is known as an alternative joint. Alternative splicing is a well-known phenomenon in nature, and many cases are known, see above. In some preferred embodiments, at least one splicing control sequence is associated with a heterologous open reading frame such that, in at least one variant of the splice, the heterologous open reading frame is interrupted, for example by a codon of stop or a frame offset, while in at least one alternative variant of the splice the heterologous open reading frame is not interrupted. Transcripts of the second type encode or potentially encode a functional protein, while those of the first type encode a protein function altered, interrupted or even no function, activity or stability relative to those of the second kind. It will generally be apparent to the person skilled in the art that the heterologous open reading frame can in itself be a composite or a fusion of sequences from several sources. The splicer to produce a functional protein can still produce an altered protein related to the heterologous prototype that opens the reading frame, for example if the alternatively spliced inserted intron includes the sequence that is exonica in all alternative splice forms, and therefore conserved in mature mRNAs of the second type. However, it is particularly preferred that at least one transcript remove all, or substantially all, alternately spliced sequence inserted, such that the heterologous open reading frame is restored, or substantially restored, to the intact form, remaining little or no endogenous sequence associated with the intron in the mature mRNA. The term endogenous is used here in contrast to heterologous, so it will be understood that this refers to a sequence which, in the native type, is normally found in association with, or is linked to, at least one element or component at least of the one splice control sequence. Alternatively, one or more transcripts can remove additional nucleotides, to interrupt the heterologous open reading frame, not by the insertion of additional nucleotides (eg stop codon or frame shift), otherwise also potentially the coding sequence that interrupts the function), but rather by deletion of nucleotides from the heterologous open reading frame, for example in order to induce a "frame shift". One or more variants of the splice may have trimmed said nucleotides, but at least one variant must retain these nucleotides, so as not to induce a frame shift in at least one variant. These removed nucleotides are those that are removed in addition to sequences that are normally unblocked, for example the intron, where an intronic sequence can be considered as one that forms part of an intron in at least one variant of alternative splicing of the natural analogue. When the exonic nucleotides must be removed, they must be removed in multiples of three, if desired to avoid a frame shift, but as a single nucleotide or multiples of two (which are not also multiples of three) if desired to induce a frame shift. It will be appreciated that if only one or certain multiples of two nucleotides are removed, then this could lead to a completely different sequence of the protein that was encoded in or around the junction of the mRNA junction. This is particularly the case in a system mode where the exons of the cartridge are used to interrupt an open reading frame in some variants but not in others of the joint, such as, for example, tra, especially Cetra.

In another preferred embodiment of the present invention, all or a portion of an open reading frame is in an exon of the cartridge, for example some modalities of Dsx derived from Aedes, are provided, for example, with a tTAV coding region. in a cartridge exon that is only present in specific splice variants to females. When the mediation of the alternative splice is specific to the splice, it is preferred that the splice variant encoding a functional protein to be expressed in an organism is the splice variant F1, ie a splice variant found only or predominant in females, and it is preferably the most abundant variant found in females, although this is not essential. Correspondingly for configurations where all or a portion of a functional open reading frame is located in an exon of the cartridge, it is preferred that this cartridge exon is included in the transcripts found only or predominantly in females, and such transcripts are preferably, individually or in the combination, the most abundant variants found in females, although this is not essential. In a preferred embodiment, the sequences are included in a hybrid or recombinant sequence or construct that are derived from the natural intronic sequences that are subject to alternative splicing, in their native or original context. Therefore, an intronic sequence can be considered as one that forms part of a model in at least one variant of alternative splicing of the natural analogue. Thus, the sequences corresponding to the individual contiguous extensions of the natural intronic sequence, but also hybrids of such sequences, including hybrids from two different natural intronic sequences, and also from sequences with contiguous extensions in relation to the deletions are foreseen. or of the individual inserts of the naturaltronic sequence, and their hybrids. Sequences derived from natural intronic sequences may be associated, in the invention, with sequences that are not part of any natural intron. If such sequences are transcribed, and are preferably preserved in the mature RNA in at least one variant of the splice, they can then be considered exonic. It will also be appreciated that reference to a "displacement" of the frame; it could also refer to the direct coding of a stop codon, which is also likely to lead to a non-functional protein as an interruption of the spliced sequence of the mRNA caused by the insertion or deletion of nucleotides. It is also envisaged the production of several variants of the splicing of two or more diverse proteins or the polypeptide sequences of differentiated function, in addition to the production of two or more diverse proteins or polypeptide sequences of which one or more have no function predicted or discernible. It also provides for the production of various variants of the splicing of two or more diverse proteins or polypeptide sequences of similar function, but differing subcellular localization, stability or binding capacity or association to other proteins or nucleic acids. Preferably, at least one splicing control sequence is intronic and comprises at its 5 'end a nucleotide of the guanine (G). In other words, the 5 'nucleotide of the splice control sequence, 3' to the splice donor site, and preferably at the interface or junction of the exon with the splice control sequence, is guanine (G), at pre-RNA, or C in an antisense sequence of the DNA that corresponds to it. In addition, the adjacent nucleotide (3 'to G) is preferably the cytosine (c) in the pre-RNA, or a corresponding G in a DNA sequence, but is more preferably the uracil (U) in the pre-RNA, or a corresponding A in an antisense sequence of the DNA. Thus, the two 5 'nucleotides of the splicing control sequence are preferably 5' GT with respect to the DNA sense strip, 5 '- GU in the primary transcript. Preferably, at least one intronic control sequence of the splice also comprises at its 3 'end a 3' guanine nucleotide and preferably AG-3 'at the junction of the acceptor site of the splice with the exon, for example, see FIG. 34. Preferably, the 5 'flanking sequence to the splice donor site in the system encompasses 5'-TG, in order to represent the sequence 5'-TG- * - splicing control sequence ** - 3 ', where * represents the splice donor site and ** represents the site of the splice acceptor. Preferably, the splicing control sequence is also flanked at its 3 'end by a nucleotide G, and more preferably by the GT nucleotides, such that the sequence could be represented as: 5' - TG- * splicing control sequence * * - GT-3 '. It will be appreciated that this is the DNA sequence of the sense strip (TG). Thus, the transcribed pre-RNA will read the UG for example, when U replaces T. The guanine or thymine derivatives having the same function are also considered. It is particularly preferred that the splice is sex specific and that it is further mediated or controlled by the binding of the TRA protein or the TRA / TRA2 protein complex, or homologs thereof. In insects, for example, the TRA protein is expressed differentiated in different sexes. Particularly, the TRA protein is known to be largely present in females and, therefore, is mediating the alternative splicing in such a way that a coding sequence is expressed in a sex-specific manner, i.e. protein is expressed in some cases only in females or in a much higher level in females than in males or, alternatively, in other cases that a protein is expressed only in males, or in a much higher level in males than in males. in females. While it is preferred that the protein be expressed only in males, however it is particularly preferred that the protein be expressed only in females. The mechanism to achieve this alternative sex-specific splicing mediated by the TRA protein or the TRA / TRA-2 complex is known and described, for example by Pane et al. (Development 129, 3715-3725 (2002)). Preferably, the at least one splicing control sequence encompasses, and preferably consists of, the intron derived from the tra gene of Ceratitis capitata (Cctrd), which has an alternately spliced region. In the F1 transcript, as illustrated in Figure 33 (Figure 2A of Pane et al (2002) supra), this is the first intron. Homologs of the tra gene in other species, such as Bactrocera oleae, Rose Ceratitis, Bactrocera zonata and Drosophila melanogaster have also alternatively spliced regions in a similar location within the tra coding sequence, introns derived from these insects are also particularly preferred. The splicing pattern in Cetra is conserved particularly well, with those transcripts found in the males containing the additional exonic material related to the F1 transcript, such that these transcripts do not encode the integral, functional protein Tra. On the contrary, the transcription of F1 encodes the integral, functional protein of Tra; this transcript is substantially specific to females in most stages of the life cycle, although it is speculated that very early embryos of both sexes may contain a small amount of this transcript. We describe the spliced sequence outside the F1 transcript, but not transcripts not specific to males or non-sex specific, such as the intron tra, or even the F1 intron. Thus the version of this sequence found in the Cetra gene is the Cetra intron. Thus the tra gene is regulated in part by the sex-specific alternative splice, while its key product, the Tra protein, is properly involved in the alternative splicing. In insects, the sex-specific alternative splicing mediated by the TRA protein, or a complex spanning the TRA and TRA2 proteins, includes the splice control sequences derived from the doublesex gene (dsx) and also from the intron tra, although this would exclude the Drosophila intron (Dmtra), which is mediated mainly by the product of the Sxl gene in Drosophila, instead of the TRA or the TRA / TRA2 complex. In addition to Drosophila, the product of the differentiated Sxl gene is not expressed in the different sexes. It is not believed that Sxl acted in the mediation of alternative sex-specific splicing in non-drosophilid insects. Examples of the TRA protein that binds to protein binding sites (recognized nucleotide sequences specifically by the TRA protein) in the intron are preferably diptera, preferably of the family Tephritidae, preferably of the genera Ceratitis, Anastrepha or Bactrocera. However, it is also considered that the other diptera, such as drosophilides or mosquitoes of the various forms discussed below, is also capable of providing the TRA protein or its homologs which are capable of binding to the appropriate sites in the control sequences. of the splice derived from the dsx gene, the tra gene or the tra intron, that is to say the intron tra alternatively spliced completely removed in the F1 transcript, even in those cases, such as Drosophila, where the natural tra gene (Dmtra) is not regulated by the TRA protein. In some modalities, the "intron" of the tra; it can be defined as a splicing control sequence in which the alternative splicing of RNA transcription is regulated by TRA, for example by binding it, alone or in combination (ie when complexed) with TRA2. This excludes the intron of the Drosophila tra. It is particularly preferred that the splicing control sequences be derived from the intron tra. The intron can be derived, as described elsewhere, from Ceratitis, Anastrepha or Bactrocera. The intron of Ceratitis capitata of the transforming gene was initially characterized by Pane et al. (2002), supra. However, it will be appreciated that homologs exist in the other species, and can be easily identified in the species mentioned and also in its various genera. Thus, when reference is made to the tra it will be appreciated that it is also related to tra homologs in other species, especially in the species of Ceratitis, Anastrepha or Bactrocera. By the term "derivative" it will be understood that, referring to the intron of the tra, it refers to the sequences that closely approximate or replicate the intron of the tra, as described in the art, in this case by Pane et al. (2002) , supra. However, it will be appreciated that, since these are the intronic sequences, some nucleotides can be added or deleted or replaced without a substantial loss in their function. Preferred examples include the dsx intron, preferably provided in the form of a minigene. In this case, it may be preferable to the deletion, as we have done in the examples, important quantities of alternatively spliced introns, for example in some cases 90% or more of an intron, still retaining the function of alternative splicing. Thus, although large deletions are envisaged, it is also envisaged that smaller, and even individual, insertions, substitutions or deletions of the nucleotide are also preferred. The exact length of the splicing control sequence derived from the intron is not essential, as long as it is able to mediate alternative splicing. In this regard, it is thought that around 55 to 60 nucleotides are the minimum length for a modified tra intron, although the native intron type of the tra - (F1 splice variant) of C capitata, is in the region of 1345 nucleotides long. It is particularly preferred that the integral sequence 1345 of the Cetra ntd be used. As with all nucleotide sequences described herein, it is preferred that some degree of sequence homology be considered, unless otherwise evident. Thus, it is preferred that the splicing control sequence have at least 80% sequence homology with reference to SEQ ID No., preferably 80% homology with reference to SEQ ID No., preferably at least 80% homology with reference to SEQ ID No., more preferably at least 90% homology with reference to SEQ ID No., preferably at least 95% homology with reference to SEQ ID No., more preferably at least 99% homology with reference to SEQ ID No., and more preferably at least 99.9% homology with reference to SEQ ID No .. A convenient algorithm such as BLAST can be used to determine the homology of the sequence. If large quantities of sequence are deleted cf of the native type, then the comparison of the sequence can be performed over the entire length of the native type or over the aligned sequences of similar homology. However, it will be understood that despite the homology before said sequence, certain elements must be conserved, particularly the nucleotides and the site of the flanking branch, for the efficient operation of the system. That is, while the portions may be suppressed or altered in another way, alternative splicing functionality or activity must be maintained, of at least 30%, preferably 50%, preferably 70%, preferably 90%, and more preferably 95% compared to the native type. This could be the high cf of the native type, also, by conveniently manipulating, for example, the binding sites of the alternative splicing factors or interacting with the spliceosome. Particularly, it is preferred that when the splice control sequence encompasses a modified TRA intron, it encompasses at least 20 to 40 base pairs from 5 'and, preferably, the 3' end of such an intron. In addition, it is preferred that at least 3 or 4 and more preferably, at least 5, preferably 6, preferably 7 and more preferably the 8 of the 8 mandatory domains attributed to TRA of the C. capitata intron be provided, as required. taught by Pane and others. (2002), or its counterparts. Of course, more of these sites are discovered in due time, so it is considered that the splicing control sequence could include more than 8 sites. In fact, it is considered that the more than 8 sites can be manipulated in the splicing control sequence and that the splice of the alternative can be regulated in this way, especially if some sites are linked with different affinities that lead to different results of alternative splicing. A consensus sequence for the assumed mandatory domains of TRA of the intron tra C. capitata, is given below as SEQ ID NO 1, a DNA sequence, although the corresponding equivalent of the RNA is also preferred. Preferred consensus sequences are 1: TCWWCRATCAACA (SEQ ID No. 1), where W = A or T and R = A or G. Similar considerations apply to doublesex, where the consensus sequence for the TRA protein is also the same. given under SEQ No. 1, as a protein complex comprising Tra proteins and TRA2 is a key regulator of doublesex alternative splicing, as it is for tra homologs (however not the tra homologs found in drosophilides). As mentioned above, the splicing control sequences are preferably derived from the intron, preferably from the Tephritidae family. It is particularly preferred that the intron is derived from B. zonata or, preferably, from other non-drosophilid fruit flies. However, it is particularly preferred that the intron be derived from the genus Ceratitis, particularly C. rosa and, more preferably • C. capitata. They are more widely known as the fruit flies of Natal and the Mediterranean, respectively.

With respect to the intron derived from B. zonata, we have shown that this can lead to the alternative specific connection to sex in the transgenic Mexican fly (of Anastrapha ludens) and in the transgenic Mediterranean fly (C. capitata). We have also shown that a variety of proteins can be expressed in a sex-specific manner by means of alternative splicing, including tTAV 3 and Rpr. Regarding the intron derived from C. rosa, we have successfully provided alternative splicing in a specific manner to each sex of a transgene in the Mediterranean fly. With respect to the intron derived from C capitata (Mediterranean fly), we have shown that it can mediate the specific connection to each sex in the transgenic Mediterranean fly, and other tefritids, and the other tephritida such as A. ludens (mex fly). . Not only that, we have shown that this intron can work successfully through a range of insects and, particularly, Diptera. In fact, we have shown that the TRA intron of C capitata (designated as Cetra) can provide sex-specific splicing in the transgenic Drosophila, which is not a tefritida, and also in the Aedes aegypti mosquito. Although mosquitoes are dipterous, they were derived from Drosophila and Tefritida about 250 million years ago and, therefore, are related much more distantly than drosophilides are from tefritides, for which the Time of divergence has been estimated at 120-150 million years. Thus, this demonstrates the broad applicability of the present invention through a wide range of insects. With respect to splicing control sequences derived from the dsx intron, we have also shown that this can be used to alternatively splice, in a sex-specific manner, into a wide range of insects. Accordingly, it is particularly preferred that the dsx be derived from Bombyx mori (silk moth), Pectinophora gossypiella (pink worm) Pectinophora gossypiella, Cydia pomonella (apple moth), Drosophila, and mosquitoes such as Anopheles sp. Example A. gambiae . Particularly preferred mosquitoes include Stegomyia spp., Particularly S. aegypti. (also known as Aedes aegypti). In fact, in A aegypti, we have demonstrated a considerable number of DNA constructs, which are capable of providing alternative splices specific to sex. It will be appreciated that the system or construct is preferably administered as a plasmid, but is generally tested after integration into the genome. The administration can be carried out by methods known in the art, such as parenteral, intravenous, intramuscular, transdermal, oral administration through a mucous membrane, and so on. Embryo injection is particularly preferred. The plasmid can be linearized before or during administration, and not all the plasmid can be integrated into the genome. When only part of the plasmid is integrated into the genome, it is preferred that this part includes at least the splicing control sequence capable of mediating the alternative splicing. Preferably, the expression system of the polynucleotide is a recombinant dominant lethal genetic system, the lethal effect thereof being conditional. Suitable conditions include temperature, such that the system is expressed at a temperature but not, or to a lesser degree, at another temperature, for example. The lethal genetic system can act on specific cells or tissues or impose their effect on the whole organism in general. Systems are also provided that are not strictly lethal but substantially harm health, for example they lead to blindness, impediment to flying (for organisms that could fly normally), or sterility. Systems are also provided that interfere with the determination of sex, for example that transform or tend to transform totally or partially an organism from one sexual type to another. It will be understood that all such systems and consequences are encompassed by the lethal term as used herein. Similarly, the term "kill", and similar terms refer to the effective expression of the lethal system and thus to the imposition of a harmful or sex-distorting phenotype, for example death.

Preferably, the polynucleotide expression system is a recombinant dominant lethal genetic system, the lethal effect which is conditional and is not expressed under permissive conditions requires the presence of a substance that is absent in the natural environment of the organism, in such a way that the lethal effect of the lethal system occurs in the natural environment of the organism In other words the coding sequences encode a lethal bound to a system such as the tet system described in WO 01/39599 and / or WO2005 / 012534. It is in fact preferred that the expression of the lethal gene in question be under the control of a repressible trans-activator protein. It is also preferred that the gene whose expression is regulated by the alternative splicing encodes a transactivator protein such as tTA. This is not incompatible with the regulated lethal protein. In fact, it is particularly preferred that it be both. In this regard, we particularly prefer that the system include a positive regeneration system as taught in WO2005 / 012534. Preferably, the lethal effect of the dominant lethal system is conditionally suppressible. Suitable organisms under which the current system can be used include mammals such as mice, rats and farm animals. Fish are also preferred, such as salmon and trout. The plants also they prefer, but it is particularly preferred that the host organism be an insect, preferably a diphtheria or a tephritida. Preferably, the organism is not a human being, preferably it is not a mammal, preferably it is not a bird, preferably it is an invertebrate, preferably it is an arthropod. In particular, it is preferred that the insect be of the order Diptera, especially upper Diptera and particularly that it be a Tefiritida fruit fly, preferably Mediterranean fly (Ceratitis capitata), preferably Mexican fly (Anastrepha ludens), preferably Oriental fruit fly (Bactrocera) dorsalis), olive fly (Bactrocera oleae), melon fly (Bactrocera cucurbitae), Natal fly fruit fly (Pink Ceratitis), cherry fly (Rhagoletis cerasi), Queensland fruit fly (Bactrocera tyron 'i), peach fruit fly (Bactrocera zonata) Caribbean fruit fly (Anastrepha suspensa) or fruit fly of the West Indies (Anastrepha obliqua). It is also particularly preferred that the organism be a mosquito, preferably of the genus Stegomyia, Aedes, Anopheles or Culex. Particularly preferred are Stegomyia aegyptae, also known as Aedes aegypti, Stegomyia albopicta (also known as Aedes albopictus), Anopheles stephensi, Anopheles albimanus and Anopheles gambiae. Among the dipterans, another preferred group is Calliphoridae, particularly the New World worm (Cochliomyia hominivorax), the old world worm (Chrysomya bezziana) and the Australian fly of the wool cattle (Lucilia cuprina). Lepidoptera and Coleoptera are also preferred, especially moths are especially preferred, including apple moth (Cydia pomonella), and silkworm (Bombyx mori), pink worm (Pectinophora gossypiella), diamond moth (Plutella xylostella), the gypsy moth (Lymantria dispar), the orange worm (Amyelois transitella), peach tree borer (Anarsia lineatella) and the rice stem borer (Tryporyza incertulas), also the nocturnal moths, especially Heliothinae. Among the coleoptera, the Japanese beetle (Popilla japonica), striped beetle (Graphognatus spp.), Weevil (Anthonomous grandis), corn rootworm (Diabrotica spp) and Colorado potato worm (Leptinotarsa decemlineata). Preferably, the insect is not a drosfilide, especially Dm. Thus, in some embodiments, expression in Drosophilides, especially Dm, is excluded. In other embodiments, the splicing control sequence is not derived from the intron of a drosfilide, especially Dm tra. It is preferred that the expression of the heterologous sequence of the polynucleotide leads to a phenotypic consequence in the organism. It is particularly preferred that the functional protein is not beta-galactosidase, but can be asated with visible markers (including fluorescence), viability, fertility, fertility, fitness, ability to fly, vision, and behavioral differences. It will be appreciated, of course, that, in some embodiments, the systems of expression are typically conditional, with the phenotype expressed only under some conditions, for example restrictive. In another aspect, a method of population control of an organism in a natural environment is also provided, which consists in: i) raising a stock of the organism, the organism carrying a gene expression system that spans a system according to the present invention that is a dominant lethal genetic system, ii) distribute reserve animals in the environment in an area for population control; and iii) achieve control of the population through lethality in the early stages by means of the expression of the lethal system in the descendant resulting from the crossing of the individuals of that reserve with individuals of the opposite sex of the native population. Preferably, the lethality at an early age is embryonic or before sexual maturity, preferably early in development, more preferably in the early life stages or embryonic stages of life. Preferably, the lethal effect of the lethal system is conditional and occurs in the natural environment by means of the expression of a lethal gene, the expression of the lethal gene is under the control of a repressible trans-activator protein, the rearing that is under permissive conditions in the presence of a substance, the substance is not present in the natural environment and is capable of repressing the transactivator. Preferably, the lethal effect is expressed in the embryos of the descendants said. Preferably, the organism is an invertebrate multicellular animal or as described elsewhere. A method of biological control is also provided, which includes: i) the rearing of a stock of male and female organisms transformed with the expression system according to the present invention under permissive conditions, allowing the survival of males and females, for provide a dual sex biological control agent; ii) optionally before the next step that imposes or allows the restrictive conditions to cause the death of individuals of one sex and providing a biological control agent of a single sex that includes individuals of the other sex carrying the conditional lethal genetic system; Ii) freeing the biological control agent of dual sex or of a single sex in the environment in a specific place for biological control; and iv) achieve biological control with the expression of the system genetic in descendants resulting from the crossing of the individuals of the biological control agent with the individuals of the opposite sex of the native population. Preferably, there is sex separation before distribution of the organism by the expression of a sex-specific lethal genetic system. Preferably, the lethal effect results in a kill greater than 90% of the target class of the progeny of crosses between the released organisms and the wild population. A method of sex separation is also provided, which includes: i) raising a stock of male and female organisms transformed with the genetic expression system under permissive or restrictive conditions, allowing the survival of males and females, and ii) eliminating permissive or restrictive conditions to induce the lethal effect of the lethal gene in one sex and not in the other by means of the alternative connection specific to the sex of the lethal gene. Preferably, the lethal effect results in the killing of more than 90% of the progeny target class of couplings between the released organisms and the wild population. A method for biological or population control is also provided; i) breeding a reserve of male and female organisms transformed with the genetic expression system under permissive or restrictive conditions, allowing the survival of males and females; ii) eliminate the permissive or restrictive conditions to induce the lethal effect of the lethal gene in one sex and not in the other by alternative, sex-specific splicing of the lethal gene that produces sex separation; iii) sterilize or partially sterilize the separated individuals and iv) obtain control through the release of sterile or partially sterile individuals separated within the natural environment of the organism. Preferably, the sterilization will be carried out with the use of ionizing radiation. However, radiation avoidance methods such as those used in the Sterile Insect Technique (SIT) are generally preferred and have many cost and health advantages over methods associated with or followed by the use of radiation. A method to selectively remove females from a population is also provided. The equivalent for males is also considered. Methods of sex separation are enormously important commercially in, for example, silkworms, where males produce more and better silk than females. Thus, methods of sex separation that eliminate females and, in particular, female silkworms are particularly preferred. It is also considered that the functional protein can be expressed differentially, but can be detected in more than one variant of the splice and preferably, therefore, in both sexes, for example. Such examples include a fluorescent protein, such as eGFP, CopGFP and DsRed2. This can be used in a method of safe separation or classification of sex, so that one can separate the two types without killing any of them. We have also surprisingly discovered that the placement of the splicing control sequence can be altered and we have obtained better results. Preferably, the splicing control sequence is the "first" splicing control sequence, when read from the promoter, in the 5 'to 3' direction. We have found that in certain constructs with an intron in the 5 'UTR of the system leading to reduced levels or expression of the alternatively spliced protein mediated by the splicing control sequence of the present invention. Preferably, the splicing control sequence is 3 'to the start codon. Preferably, the splicing control sequence is inserted into the first exon, i.e. the stretching of the sequence immediately 3 'to the site of the start of transcription. It will be understood that such terms may refer to the DNA sequence encoding the transcription, or to the transcription of the RNA itself. When the splicing control sequence is 3 'to the start codon, it is preferred that it also be 5' to the first stop codon in the frame (which is 3 'a and in the frame with the start codon), so that the Alternative splicing produces transcripts that encode that encode various protein or polypeptide sequences. Thus in a preferred embodiment, the construction or sequence of the polynucleotide encompasses the following elements in the order 5 'to 3', with respect to the sense strip or the primary transcript: start of transcription, start of translation, capable intron of alternative splicing, coding sequence for all or a part of a protein, stop codon. The splicing control sequence can be defined as preferably up to and including the 5 'G (GT / C) and its 3'G equivalent, especially in tra, but as mentioned above, this may include a certain exonic sequence and therefore therefore, it could include the nucleotide closest to 3 '(last) of the exon (ie G). It is particularly preferred that the splicing control sequence be immediately adjacent, in the 3 'direction, the start codon, so that the G of the ATG is 5' at the start (5 'end) of the control sequence of the splice. This is particularly advantageous since it allows the G of the ATG start codon to be the 5 'flanking sequence to the splice control sequence.

Alternatively, the splicing control sequence is 3 'to the start codon but within 1000 bp exons, preferably 500 bp exons, preferably 300 bp exons, preferably 200 bp exons, preferably 150, preferably bp exons 100 bp exons, more preferably exogenous bp, more preferably 50 ex exogenous bp, more preferably ex exogenous 30 bp, more preferably 20, and most preferably 10 or even 5, 4, 3, 2, or 1 bp exon. The present invention is an improvement in the system defined as LA1188 in WO2005 / 012534. This plasmid had several defects, the main one of which is that the exonic nucleotides were deleted with the Cetra intron used here, in such a way that an induced shift in transcription is resulted. Specifically, in addition to the sequence derived from Cetra (the Cetra intron), 4 nucleotides of the tTAV sequence were removed in the transcript specific to the females. Therefore, although several alternately spliced transcripts were produced, including a transcript specific for females, none was able to encode the functional protein tTAV. Therefore, this construct was not able to provide the sex-specific expression of the functional protein tTAV. Since the splice was not directed to the splice donor sequence (5'-GT ...) normally used in the Cetra intron, this construct clearly did not contain all the sequences regulators necessary to direct the splice in the form of Cetra's intron in "its native context". However, this is another problem. Probably the only missing was the flanking TG ... GT, of which it is possible that only the 5 'G would matter. A key benefit of the present invention, particularly as regards tra, that the requirements for the exonic sequence are so minimal (for example 2 nucleotides at each end) that can be easily designed in most coding sequences, using redundancy in the genetic code. In such a way that the "extra" exonic nucleotides can be both part of the heterologous sequence of the protein, and at the same time the sequence that flanks the intron in its native context. In addition, the Ccfra intron in LAI 188 was + 132bp 3 'to the G of the ATG start codon (to the last exonic nucleotide). In fact, although the Cetra intron in LA1188 is the first intron read in the 5 'to 3 direction from the ATG start codon, it is not the "first" intron when read in the 5' to 3 'direction from the promoter . In fact, it is the 2nd intron, as there is another intron (derived from the Drosophila melanogaster Adh gene) upstream from the ATG start codon. This information is included in table 3. It will be understood that when reference is made to the ATG start codons or to flanking G, or 5'-TG ... GT-3 'sequences this in relation to a DNA sequence, but it also covers the corresponding DNA antisense sequence and, likewise, the corresponding RNA sequence.

Description of the sequences of the present invention SEQ ID NO. 1 consensus sequence tra SEQ ID NO. 2 flanking sequence of 5 'LA3097 SEQ ED NO. 3 3 'flanking sequence LA3097 SEQ ID NO.4 primer 688 - el-transcr SEQ ID NO. 5 primer 790 - Aedsx-m-r2 SEQ ID NO. 6 primer 761 - Aedsx-fem-r SEQ ID NO.7 primer AedsxRI SEQ ID NO. 8 consensus sequence of Pane and others SEQ ID NO. 9 consensus sequence of Scali and others 2005 SEQ ID NOS. 10-33 and 107-138 consensus sequences of binding sites assigned Tra / Tra2 deduced from Drosophila (see Table 2). 'SEQ ID NO. 34: Open reading frame of tTAV SEQ ID NO. 35: tTAV protein sequence SEQ ID NO. 36: Open reading frame of tTAV2 SEQ ID NO. 37: Protein sequence of tTAV2 SEQ ID NO. 38: Open reading frame of tTAV3 SEQ ID NO. 39: Protein sequence of tTAV3 SEQ ID NO.40: Fragment 1 of the sequence specific to the female pink worm dsx SEQ ID NO.41: Fragment 1 of the specific sequence to the female pink worm dsx (PBW, Pectinophora gossypiella) SEQ ID NO .42: Fragment of the sequence specific to the male pink worm dsx (PBW, Pectinophora gossypiella) SEQ ID NO.43: Partial genetic sequence of Aedes aegypti dsx. the AU exonic sequence is included, but only the partial intron sequence - see figures 47 and 48 for the annotation. SEQ ID NO.44: Female genetic sequence dsx of apple moth (Cydia pomonella): includes an extension of unknown nucleotides, preferably less than 100, preferably less than 5.0, more preferably less than 20, more preferably less than 10 , and most preferably less than 5. SEQ ID NO.45: Male dsx sequence of apple moth (Cydia pomonella). SEQ ID NO.46: Sequence of the construction / plasmid of pLA3435-Bombyx mori-dsx. SEQ ID NO.47: Sequence of the construction of pLA3359-Anopheles gambiae dsx. SEQ ID NO.48: Sequence of the construction of pLA3433-Agdsx (Anopheles gambiae) including 2 exons. SEQ ID NO.49: Sequence of intron construction pLAI 188-cctra. SEQ ID NO. 50: Sequence of the construction of pLA3077-a Cetra intron-tTAV. SEQ ID NO. 51: Sequence of the construction of pLA3097-a Cetra intron-tTAV. SEQ ID NO. 52: Sequence of the construction of pLA3233-Cctra-intron-tTAV2.

SEQ ID NO 53: Sequence of the construction of pLA3014-Cctra-intron-Ubiquitin-reaperKR. SEQ ID NO. 54: Construction sequence of pLA3166-Cctra intron-Ubiquitin-reaperKR. SEQ BD NO. 55: Sequence of pLA3376-Bztra intron-reaperKR and Bztra-intron-tTAV3. SEQ ED NO. 56: Construction sequence of pLA3242-Crtra intron-reaperKR. SEQ ID NO. 57: Partial sequence of a transcript of a male generated in Drosophila melanogaster of the LA3077 transformants that differs from the sequence generated in the lineages of the Mediterranean fly LA3077 lines. This sequence corresponds to the M3 transcript shown in Figure 36. SEQ ID NO. 58: Partial sequence of a homologo of Bactrocera zonata tra. Intronic sequence that was predicted was going to be unpacked in a specific transcript for the females of B. zonata tra (+ 3 to + 970bp in the sequence). The flanking exonic nucleotides are found in positions 1-2 and 971-972, this is at the 5 'and 3' ends of the intronic sequence. In fact it is worth noting that the intronic sequence is flanked at its 5 'end by a guanine nucleotide, which is believed to be critical for an extraction for clean exit of the intron. SEQ ID NO 59: Partial sequence of a homologue of Ceratitis rosa tra. Intronic sequence predicted to be unpacked in a specific transcript for females of C. rosa tra (+ 3 to 1311 bp of sequence). Flanking exonic nucleotides are present in positions 1-2 and 1312-3. Again it is worth noting that the intronic sequence is flanked at its 5 'end by a guanine nucleotide, which is believed to be critical for an extraction for clean exit of the intron. SEQ ID NOS. 60-70: Primers referred to in Figures 44-46 and 50-51. SEQ ID NO. 71: Fragment 3 of the sequence specific to the female pink worm dsx (PBW, Pectinophora gossypiella) SEQ ID NO. 72: Open reading frame of ubiquitin from Drosophila melanogaster. SEQ ID NO. 73: Ubiquitin protein sequence of Drosophila melanogaster. SEQ ID NOS. 74-105 Are the primers described in the examples SEQ ID NO. 106 is nucleotide sequence LAI 172, which includes a plasmid base. SEQ ID NOs 107-138 are as described above. SEQ ID NO. 139 HSP primer SEQ ID NO. 151 complete plasmid LA3619 SEQ ID NO. 140 sequence of primers VP 16 SEQ ID NO. 141 Primer AgexonlF SEQ ID NO. 152 complete plasmid LA3612 SEQ ID NO. 142 sequence of primers TETRRI SEQ ID NO. 143 sequence of plasmids LA3576 SEQ ID NO. 153 plasmid sequence LA3491 SEQ ID NO. 144 sequence of plasmids LA3582 SEQ ID NO. 154 sequence of plasmids LA3515 SEQ ID NO. 145 sequence of plasmids LA3596 SEQ ID NO. 155 sequence of plasmids LA3545 SEQ ID NO. 146 PBW-dsx (Fig 6) SEQ ID NO. 156 plasmid sequence LA3604 SEQ ID NO. 147 bombyx-dsx (Fig 6) SEQ ID NO. 157 sequence of plasmids LA3646 SEQ ID NO. 148 codling-dsx (Fig 6) SEQ ID NO. 158 sequence of plasmids LA3054 SEQ ID NO. 149 DSX Minigen of construction LA3491 SEQ 3D NO. 159 sequence of plasmids LA3056 SEQ ID NO. 160 plasmid sequence LA3488 SEQ ID NO. 150 DSX Minigen2 of construction LA353 SEQ ID NO. 161 sequence of plasmids LA3641 SEQ ID NO. 162 LA3570 plasmid sequence The invention will now be described by reference to the following, non-limiting examples. EXAMPLES Transformer Example 1 - Intron of Ceratitis capitata We have prepared an insertion of a Cetra intron cartridge in an open synthetic reading frame (ORP). Two versions of this correctly butt in the Mediterranean fly, that is to say the splice of the intron cartridge of Cetra They faithfully recapitulate what they would normally do in the context of the endogenous Cetra gene. This is to produce 3 variants of the splice (main or only) in the females, one of which (called F1) is specific to the females while the other two are found in both males and females (called M1 and M2). Since each of the non-specific transcripts contains extra exonic material with stop codons, we have also arranged this so that only the female variant of the splice produces functional protein. Each of these constructions (LA3077 and LA3097) has the Cetra intron flanked by TG and GT (to give 5 '.TG | i ntron | GT ... 3'.) An older construction, which does not work perfectly, is LA1188 LA1188 is absolutely well characterized - the splice is exactly like the previous one except that the 4 additional nucleotides are removed.The intron is in the context 5 '... TGGCAC | intron] GT ... 3'; the splice removes the 4 additional bases, ie 5 '... TG | GCACintron | GT ... 3' (figure 33) In all cases the intron is invariant, and is simply the complete sequence of the Cetra intron. introns, starts in GT and ends in AG.Almost all introns start in GT, so the use of the rare alternative GC in LA1188 is surprising [the GC-AG introns are a known alternative - in a large-scale study, reported that 0.5% of all introns used GC-AG (Burset et al., 2001), although this can be a disadvantage, particularly for alternatively spliced introns, of which maybe 5% could use GC-AG (Thanaraj and Clark, 2001)]. The RT-PCR analysis was performed in LA3077, (a construct of positive feedback with the CcTRA intron in the open reading frame tTAV). Transformed adult flies of both sexes were reared on a diet substantially free of tetracycline ("without tetracycline") for 7 days. The flies were then harvested for RNA extraction and RT PCR using the primers (HSP-SEQ ID NO: 104 and VP16 SEQ ID NO: 105) to analyze the splice pattern of the CcTRA intron (Figure 34). In two female samples we found the correct pattern of the Cetra junction (776bp, corresponding to the exact removal of the Cetra intron) and we did not see any such band in the males. We found that LA3077 and LA3097 correspondingly gave a specific lethality to the repressible females. L A3077 was phenotypically tested by crossing heterozygous flies for LA3077 to the native type, with intervals of tetracycline administration. The feminine lethality extended from 50 to 70%. LA3097 (a modified version of LA3077 whereby the Cetra intron immediately follows the start codon in the tTAV ORF), demonstrated a much higher female-specific lethality level, reached 100% (figure 35). The Cetra intron was also inserted into tTAV2 in the same position LA3097, in construction LA3233, and this gave a phenotypic result similar to LA3097 (figure 35). We have also prepared transformants of LA3077 in Drosophila. Phenotypically, the construction works perfectly, which means that it is highly lethal to females. However, the sequence of the splice variants of one of these inserts has shown that the splicing of this construction in Drosophila is not quite equal to that of the Mediterranean fly (SEQ ID No. 57). The critical transcription, specific to females, is the same in both, but at least one of the non-sex-specific transcripts is different. It still incorporates an additional exonic sequence, with stop codons, but the junctions of the junction are not quite the same (figure 36). This observation is extremely important because it shows that this method (regulation of gene expression by means of alternatively spliced introns) can be used across a wide phylogenetic range. A simple test to determine whether an exonic splicing regulator up to now not characterized (such as reinforcers and suppressors) can modify the function of the alternatively spliced intron, could include the realization of the construction and the introduction of it into a target tissue, then the Examination of your splice pattern. In many cases this will not require the transformation of the germ line, so the test can be Absolutely fast, for example by transient expression in convenient tissue culture cells or in vivo. For example, in vivo testing in insects could be obtained by providing the ANA by microinjection. However, as the person skilled in the art will appreciate, microinjection along with electroporation, or electroporation, chemical transformation, ballistic methods, for example, all have been used in various contexts and such methods of introducing and expressing the protein of the plasmids are known in the art. We have also recently produced and have obtained transgenics with the Cetra intron in a different gene (LA3014) (all the above examples are in tTAV). LA3014 contains a fusion of ubiquitin-reaperKR downstream of a Cetra intron. The phenotypic data (figure 35) demonstrate that the transgenic Mediterranean fly LA3014 produced the lethality specific to the repressible females. The RT-PCR analysis in the RNA extracted from males and from adult females reared without tetracycline, using the primers (HSP, SEQ ID NO 74) and ReaperKR (SEQ ID No. 75), demonstrates that the correct splicing occurred in the females (band 508bp) and no such band was found in the males (figure 37). LA3166 is another construct with the Cetra intron placed within the coding region of ubiquitin fused to reaperKR, but put into a different position in ubiquitin. LA3166 also produces a specific lethal effect on females repressible dominant in the Mediterranean fly (figure 35). Also recently, we have produced and obtained transgenic with constructions of 'single introns' based on Cetra with the intron in a different gene (all the aforementioned examples are in the tTAV or one of its variants, ie tTAV2 or tTAV3). These constructions work according to the aforementioned. This is an important result, demonstrating that there are no essential exonic sequences in Cetra that we have simply duplicated by chance (in function, if not necessarily in sequence), in tTAV. We also have constructions of ubirpr01 of this type (LA3014 and LA3166), which also validate the ubiquitin fusion method described above.

To demonstrate the phylogenetic range of the Cetra intron we generated transgenic LA3097 and LA3233 from Anastrepha ludens. LA3097 and LA3233 were selected for injection in the Anastrepha ludens as they demonstrated the best female-specific lethality of Ceratitis capitata (see example 13). The phenotypic data were generated for 4 independent lines of LA3097 and 1 line of LA3233 (see Figure 38). Female specific lethality was generally somewhat lower in Anastrepha ludens when compared to C. capitata but reached 100% in one line. Anastrepha ludens transformed with LA3097 and reared with tetracycline until hatching were isolated and kept under tetracycline for 7 days. The RNA was then extracted and the RT-PCR analysis was performed using the primers HSP (SEQ ID No. 76) and TETRRI (SEQ ID No. 77). The correct pattern of splicing specific to females (F1 type) was observed in RNA isolates from females (348bp) but not from males, demonstrating the function of the Cetra intron in a different species (figure 39). The brightest male band and the specific female band were purified and rushed to be sequenced. We found that the female specific transcript was correctly spliced in the Mex fly females as expected for LA3097: LA3097. AGCCACCATG | GT ... AG of the intron ... | GTCAGCCGCC. The two flanking sequences indicated above are SEQ ID No. 2 and 3, respectively. Example 2: Intron tra of Bactocera zonata We isolated the intron from Bactocera zonata (B. zonata) (SEQ ID NO 58) using primers ROSA1 (SEQ ID NO.78), ROSA2 (SEQ ID NO.79), and ROSA3 ( SEQ ID NO 80). These sequences of the primers were designed based on the conserved coding sequence of tra homologs of Ceratitis capitata and Bactrocera oleae. Using ROSA2 and ROSA3 or ROSAI and ROSA3 as primers, the intron tra and its flanking coding region were amplified from Bactrocera zonata genomic DNA. After we use these PCR products as template and we amplified the fragment of the intron to make the construction -LA3376 (figure 31 and SEQ ID No. 55). The primers (BZNHE SEQ ID No. 81 and BZR-SEQ ID No. 82 of) were used to make the constructions; these primers contain the additional sequences for the purposes of reproduction. The Bztra intron in LA3376 is reproduced in tTAV3 ORF and also reaperKR. Transformants of the Mediterranean fly were generated and RNA was extracted from male and female flies. Then RT-PCR was performed in the reaperKR (SEQ ID No. 83 of HB and SEQ No. 84 of reaperKR) and splicing tTAV3 (SEQ ID No. 85) and SRY SEQ ID No. 85 and AV3F SEQ ID No. 86 ). The expected fragments of 200bp for reaperKR and 670bp for tTAV3, corresponding to the splicing in a pattern equivalent to the F1 transcript of Cetra (Pane et al., 2002), were generated in the females (Figure 40). Example 3: Isolation and splicing of the Rose Ceratitis intron (C. rosa, Natal fruit fly) The primers ROSA2 (SEQ ID NO.87) and ROSA3 (SEQ ID NO.88) were designed based on the sequence of conserved coding of Ceratitis capitata and Bactrocera oleae. Using ROSA2 and ROSA3 as primers, the intron tra and its flanking coding region were amplified from pink Ceratitis genomic DNA (SEQ ID NO: 59). Then we used the PCR products as a template and amplified the intron fragment for produce buildings. The primers (CRNHE-SEQ ID NO 89 and CRR SEQ ID NO 90) were used during the construction of LA3242 (SEQ ID NO: 56 and Figure 32. LA3242 contains the C. pink intron at the 5 'end of the reaperKR ORF. Ceratitis capitata embryos were injected with LA3242 DNA, the injected embryos were reared until adulthood with a diet substantially free of tetracycline.The RNA was extracted from adult males and females, this was used as a template for the RT PCR using the primers HB (SEQ ID No. 91) and ReaperKR (SEQ ID No. 92) The specific band of the female of the junction (200bp) expected, corresponding to the splicing in the pattern equivalent to that of the F1 transcript of Cetra, was observed in females and not in males (figure 41) Doublesex (Double sex) Example 4: Bombyx morí dsx in PBW The sequence of a homolog of the Bombyx morí (silk moth) of Drosophila Dsx (Bmdsx) has been previously described and a specific splicing product has been identified females and male (Suzuki et al., 2001). Males and females use the same 3 'polyA, and there are two female specific exons. A document has suggested that sex-specific splicing is not dependent on tra / tra2, that is, although the pattern seems the same, the underlying mechanism may be different (Suzuki et al., 2001), although its data, mainly lack of data points Recognizable link tra-tra2, however, are not required. Further, a construction of the mini-gen dsx of B mori. (which contains exonica sequence and truncated intronic sequence) has been transformed into B. mori and the germline transformants show sex-specific splicing (Funaguma et al., 2005). We have generated a Bmdsx minigene based on the sequence used in Funaguma and others, with some significant changes, and injected into the moth's pink worm to see if one can obtain sex-specific splices in a divergent species. The construction of the mini-gene that we generate does not include exon 1, which is present in males and females. In addition, we removed the intron between exon 3 and 4 (the two female specific exons), including a heterologous sequence (containing the multiple sites of reproduction, MCS), we used the reinforcement / promoter sequence Hr5-IE1 of the baculovirus AcNPV and we used a transcription sequence of the 3 'ending derived from SV40 (see figure 42 for a schematic diagram). The individual exon / flanking intron fragments used were amplified and recombined together by PCR and ligated into a construct carrying a reinforcement promoter fragment Hr5 / IE1 and SV40 3 'UTR (Figure 22 and SEQ ID No. 22). LA3435 was injected into pink worm embryos (Pectinophora gossypiella). The first instar larvae were collected after 5-7 days and analyzed individually by means of RT-PCR (using the transcriptional primers SEQ ID No. 93 and SV40-RT-P2 SEQ ID No. 94) to determine if BMdsx can be subjected to splicing specific to males and females (Figure 43). Our analysis detected the male specific band (which was previously 442bp) in 4 samples (lanes 1, 2, 3 and 4) and the female specific band (predicted to be 612bp) in 1 sample (lane 5). The correct splicing of B. mori dsx in PBW demonstrates that we can achieve (we have achieved) the sex-specific expression of a heterologous sequence (here, the MCS) in a lepidopteran using an alternative splicing system. Furthermore, since this splicing system was derived from a heterologous species, this suggests that such constructions could work on a broad phylogenetic range. However, the identification of alternative splicing systems in the species of interest is also envisioned, and methods for identifying such alternative splicing systems are provided herein or are known to those skilled in the art. By providing an MCS in our example (see Figure 42), the expression of a sequence of interest, e.g., a region of coding for a protein of interest could easily be obtained by inserting the sequence. If the sequence encodes a suitable protein, a sex-specific phenotype, for example conditional lethality specifies the sex, so it could be introduced, for example in a pink worm. Example 5: Isolation of the dsx from the apple moth The dsx gene of the apple moth (Cydia pomonella) was isolated by performing 3 'RACE using the primers that were based on sequence alignments of B. oleae, B. tyroni, C. capitata, D. melanogaster, B. mori, and A. gambiae. The RNA was isolated from a male and female apple moth and a 3'RACE was made to generate the cDNA, using the primer TT7T25 (SEQ ID No. 95). PCR was performed using the dslc primers (SEQ ID No. 96) and TT7 (SEQ ID No. 97). Two rounds of subsequent PCR were performed on the product of the first PCR using the codling2a primers (SEQ ID No. 98) and TT7 (SEQ ID No. 99) and the second round PCR product using Codling2b (SEQ ID No. 100) and TT7. The isolated sequences specific for males and females share sequence similarity with homologues previously isolated from dsx (SEQ ID No. 43 for the male and SEQ ID No.42 for the female). Example 6: Isolation of PBX dsx The pink worm dsx gene was isolated by realizing 3'RACE using primers that were based on sequence alignments of B. oleae, B. tyroni, C. capitata, D. melanogaster, B. mori, and A. gambiae. The RNA was isolated from male and female apple moths and a 3'RACE was performed to generate the cDNA, using TT7T25 (sequence defined here). PCR was performed using primers Pbwdsx2 (SEQ ID No. 101) and TT7 (SEQ ID No. 102). The nested PCR was then performed on the PCR product using primers Pbwdsx3 (SEQ ID No. 103) and TT7. Three female-specific sequences were isolated: PBWdsx-FI (SEQ ID NO: 40), PBWdsx-F2 (Figure 10), and PBWdsx-F3 (SEQ ID NO 71) and a sequence specific to the males (SEQ ID NO. ). The isolated sequences specific for males and females share sequence similarity with homologues previously isolated from dsx. Example 7: dsx in Anopheles gambiae The sequence of the dsx gene of Anopheles gambiae has been previously described (Scali et al. 2005). However, when we tried to repeat the work described in the document we found that there are some differences in the splice. When we tried to repeat the amplification of the specific transcription to the females using designed primers of the mRNA sequence (access: AY903308 for the female coding sequence and AY903307 for the male coding sequence), the amplification failed. However, when Scali and his colleagues demonstrated that there was a shared exon, which had not been previously described, we designed the primers to amplify the complete transcript and dsx gene. Using these primers and the primers designed from the DNA genomic sequence (access: Gl 19611767) we find that the splicing of the female transcript is different from that described by Scali and others 2005 (figure 44). The transcript showed that the female exon was in a different position. There are several explanations for these differences, but the most likely is a certain type of strain difference from the anopheles from which we obtained the data, or that the published sequence is not from Anopheles gambiae, or there is more than one female isoform as shown for Stegomyia aegypti in Example 20. "We have also successfully used the primers, designed around our version of the dsx splice from Anopheles gambiae, which can distinguish between males and females of Anopheles gambiae (Figure 45), which provides good evidence that the system It will serve as a sex-specific splicing mechanism when it is fused to a protein of interest, such as tTAV or a killer.The Anopheles gambiae dsx gene that we have isolated from genomic DNA, which has several changes in the nucleotide sequence compared to the genomic sequence described was reproduced in LA3359 (SEQ ID No. 47) and LA3433 (SEQ ID No. 48), schematic diagrams can be found in the figures 23 and 24, respectively. Example 8: dsx in Stegomyia aegypti The splicing of the gene seems to be similar to the dsx of Anopheles gambiae (Scali and others 2005). The dsx gene of Stegomyia aegypti is illustrated schematically in Figures 47 or 48. A specific transcription to the male (M1) is produced that does not include exons 5a or 5b. Two female specific variants of the joint (F1 and F2) have the following structure; F1 covers exons 1-4, 5a, 6 and 7 but not 5b, F2 encompasses exons 1-4 and 5b (Figure 46). In addition, another transcript (C1) is present in males and females; this encompasses exons 1-4 and 7, but not exons 5a, 5b or 6. The splicing of the gene appears to be similar to the dsx of Anopheles gambiae (Scali and others 2005). The dsx gene of Stegomyia aegypti is illustrated schematically in Figure 47 or 48. Actin 4 Example 9: Actin-4 gene of Stegomyia aegypti One way to get the sex, tissue and stage specific expression of a gene of interest is to link it to the Actin-4 gene of Stegomyia aegypti (AeAct-4). This gene is expressed only in the developing flight muscles of the female of Stegomyia aegypti (Muñoz et al. 2004). They used in-situ hybridization to an RNA to detect the expression profile of AeAct-4. We have taken a fragment of the Actin-4 gene from Stegomyia aegypti, encompassing an assigned promoter region, an alternatively spliced intron, and a section of the 5 'untranslated region (UTR) and placed in front of the sequence coding for the tTAV ( figure 49) to test the sex-specific splice function when it is fused to tTAV. We integrate LA 72 into the genome of Stegomyia aegypti using piggyBac. Two independent lines were generated (lines 2 and 8). Both lines show the correct splicing of the tTAV Actin-4 gene (Figures 50 and 51). The Actin-4 promoter and the alternatively spliced intron can therefore be successfully used to provide sex-specific junctions, tissue and the stage of a gene of interest in Stegomyia aegypti. DESCRIPTION OF THE FIGURES AND LISTINGS OF THE SEQUENCE OF EXAMPLES 1-9 FIGURE 19: A use of the element of P in the generation of the expression expression specific to the germ line of a gene of interest (gene E). The insertion of the P IVS3 element and the flanking exonic sequences upstream from a ubiquitin-E gene fusion allow germline specific expression of the E gene under an active germline A promoter-active germline promoter; B - open reading frame of the element P; C - Intron P TVS3 '; D - Ubiquitin; E - Coding region for the protein of interest eg tTAV. Figure 20: Sex-specific expression using dsx. A: Intron used as intron Cetra above, but giving the specific expression to the male. A fragment of dsx (here the version of the anopheles) is inserted into a heterologous region of the coding (shaded boxes). The intron is completely removed in the males, but in the females the coding region is prematurely terminated. B: An alternative attempt at male-specific expression, in which a heterologous region of the coding is fused to a fragment of the dsx.

C: expression specific to the female: the heterologous region of the coding is inserted into the female specific exon, such as in-frame fusion to a fragment of Dsx, or with its own start and stop codons. D: Differentiated expression: designs B and C can be combined to give expression of gene a in females and b in males. Figure 21: Alternate splice specific to the sex of Cetra A: Cetra splices into females to produce three transcripts: F1, which encodes the functional protein of Tra, and M1 and M2, which do not, because they include additional exons with the codons of unemployment (compiled by Pane and others 2002). The males produce only the M1 and M2 transcripts and therefore do not produce the functional Tra protein at all. B if this intron were to function similarly in a heterologous region of coding, this would similarly allow females, but not males, to produce functional protein X. Figure 22: Diagrammatic representation of the construct / plasmid pLA3435 (SEQ ID NO. 46). Figure 23: Map of the plasmid of the dsx gene of Anopheles gambiae pLA3359 placed under the control of an Hr5-IE1 promoter to determine the splice via the transient expression. Figure 24: Gene dsx pLA3433 from Anopheles gambiae placed under the control of an Hr5-IE1 promoter, with the addition of exon 2, to determine the splice via transient expression. Figure 25: Schematic representation of the construction pLA1188. Figure 26: Schematic diagram of the construction pLA3077 Figure 27: Schematic diagram of the construction pLA3097 Figure 28: Schematic diagram of the construction pLA3233 Figure 29: Schematic diagram of the PLA3014 construction Figure 30: Schematic diagram of the construction pLA3166 Figure 31: Schematic diagram of the construction pLA3376 Figure 32: Schematic diagram of the construction pLA3242 Figure 33: Flanking sequence of Cetra Splice of the Cetra intron in LA3077 and LA3097 is exactly as it would look in the native intron of Cetra. The splice at LA1188 results in the removal of 4 additional nucleotides. In all cases the introns are flanked by 5 'TG and 3' GT exons. Figure 34: Gel showing the specific junction to the correct sex of the introns derived from CcTra (band 776bp in females) in Ceratitis capitata transformed with LA3077. Lane 1: Marker (Eurogentec's SmartLadder ™, bands of approximately 0.8, 1.0 and 1.5kb are indicated); Lanes 2 and 3: Males of Ceratitis capitata LA3077 / +; Lanes 4 and 5: Females of Ceratitis capitata LA3077 / +. Figure 35: Phenotypic data for specific constructions for females transformed in Ceratitis capitata. Column 1: The construction designation LA #, for example LA3077, LA3097, LA3233, etc., is indicated by a number, with the lines independent of the insertion referred to with letters; Columns 2 and 3: results without tetracycline (NT) for each transformed line given in total males (2) and total females (3). Columns 4 and 5: Result of tetracycline (TET) for each transformed line given in total males (4) and total females (5). Figure 36: Transcription of the intron Cetra constructions in Drosophila and Ceratitis capitata. The top line represents the DNA of the construct that contains the intron tra flanked by the desired gene (the open box). The red box represents the male specific exons. The introns are represented by full lines. The arrow on the first line represents the positions of the oligonucleotides used in the RT-PCR experiments. The bar indicates the scale of the figure. Figure 37: Gel showing the correct splicing to the females of the sequence derived from CcTRA (508bp band) in female Ceratitis capitata transformed with LA3014. Lane 1: Marker (Eurogentec's SmartLadder ™, bands of approximately 0.4 and 1.0kb are indicated); Ceratitis capitata male LA3014 / + of lane 2; Lane 4: Female of Ceratitis capitata LA3014 / +; Lanes 3 and 5: no negative control of reverse transcriptase (bottom bands, probably genomic DNA, can be seen in lanes 2 and 4). Figure 38: Phenotypic data for transgenic Anastrepha ludens transformed with LA3097 or LA3233. Column 1: The LA # construction (LA3097 or LA3233) indicated, the independent lines of the insertion are indicated by letters; Columns 2 and 3: Results without tetracycline (NT) for each transformed line given in total males (2) and total females (3). Columns 4 and 5: Results with tetracycline (TET) for each transformed line given in total males (4) and total females (5). Figure 39: Gel showing the splice specific to the correct sex of CcTRA splice (the 348bp band in females) in Anastrepha ludens transformed with LA3097. Lane 1: Marker (Eurogentec's SmartLadder ™, bands of approximately 0.4 and 1.0kb are indicated); Lanes 2, 3 and 4: Males of A. ludens LA3097 / +; Lanes 5, 6 and 7: Females of A. ludens LA3097 / +. Figure 40: Gel showing the specific connection to the correct sex of BzTRA in the reaperKR (band 200bp in females) and tTAV3 (band 670bp in females) the regions of LA3376, in Ceratitis capitata transformed with LA3376. Lane 1: Marker (SmartLadder ™ of Eurogentec, bands of approximately 0.2, 0.6 and 1.0kb are indicated) Lanes 2 and 3: Maps of C capitata LA3376 / +. tested for splicing in reaperKR; Lanes 4 and 5: Females of C. capitata LA3376 / + tested for splicing in reaperKR; Lane 6: SmartLadder ™; Lanes 7 and 8: Machos del C, capitata LA3376 / +. tested for splicing in tTAV; Lanes 9 and 10: Females of C. capitata LA3376 / + tested at the junction in tTAV; Lane 11: SmartLadder ™.

Figure 41: Gel showing the specific junction to the correct sex in CrTRA-reaperKR (200bp band in females) in Ceratitis capitata injected with LA3242. Lane 1: Marker (Eurogentec SmartLadder ™, bands of approximately 0.2, 0.6 and 1.0kb are indicated) Lanes 2-7: native type Ceratitis capitata injected with LA3242; Lane 8: SmartLadder ™; Lanes 9-14: females native to Ceratitis capitata, injected with LA3242; Lane 15: SmartLadder ™. Figure 42: Schematic representation of the constructions of the Bmdsx minigen. Two minigene constructs derived from the dsx gene of Bombyx mori are illustrated schematically, together with the alternative splice provided for these constructions (female pattern shown above the construction, male pattern below). (A) is the construction of the dsx mini-gene of Bombyx mori used by Funaguma et al., 2005) (B) is pLA3435. A and B differ from each other in several ways: (i) Exon 1 is excluded from pLA3435s (ii) the intron between the exons specific to female 3 and 4 has been removed and a short heterologous sequence has been inserted into pLA3435 (iii) ) Funaguma and others use the promoter of the promoter iel of the baculovirus BmNPV and BmA3 3 'UTR in comparison with pLA3435 using the reinforcement / promoter of hr5-IEI of baculovirus AcNPV and a 3"SV40 3' UTR. (Iv) pLA3435 usa slightly longer sequences of the intron compared to (A) (see Figure 15 for the sequence.) Two constructions of Minigen derived from the dsx gene of Bombyx mori are schematically illustrated, along with the alternative splice provided for these constructions (female pattern is shown above the construction, male pattern is shown below). Figure 43: Specific connection to the sex of the construction of the mini-gene of BMdsx in PBW. Analysis of the transient expression of pLA3435 using RT-PCR shows the presence of a 442bp fragment (lanes 1.2.3 and 4) in males and a 612bp fragment in females (lane 5), demonstrating that the mini-gene of BMdsx with a Heterologous fragment inserted between exon 3 and 4 can correctly splice into the divergent moth, PBW. The markers are SmartLadder ™ by Eurogentec; the bands of approximately 0.2, 0.4 and 0.6 kb are indicated. Figure 44: Sex-specific splice of dsx of Anopheles gambiae. The anopheles (A) show the splice that was described by Scali and others 2005. However, the RT-PCR was performed using our primers (spl-agdsx-e3 (SEQ ID No. 60) and SPL-agdsx-m (SEQ ID No. 61)) was revealed a different pattern spliced for females, represented by Anopheles (B). Figure 45: Identification of male and female Anopheles gambiae using the dsx primers. The RNA was extracted from Anopheles gambiae males and females and the transcripts of dsx were amplified by RT- PCR using the primers spl-agdsx-e3 (SEQ ID No. 62) and SPL-agdsx-m (SEQ ID No. 63); The resulting band pattern is shown in the gel above. The bands provided for the male and female transcripts are indicated by the white arrows, have been reproduced and the bands have been ordered and are identical to the predicted sequence of our version of the transcription of dsx (see SEQ ID No. 47 ( LA3359) and SEQ ID No. 48 (LA3433)). The molecular weight markers are shown in the kb (Eurogentec SmartLadder ™, the sizes are approximate). Figure 46; Identification of male and female Stegomyia aegypti using the dsx primers. The primers for the Stegomyia aegypti RT-PCR for A and B were the aedesxFI (SEQ ID No. 64) and aedesxR5 (SEQ ID No. 65) were initially tested in the pupae, a stage in the life of the Stegomyia aegypti whose dry which can be conveniently and accurately determined; The resulting amplification of RT-PCR is shown in the gel image (A). The male and female pupae a distinctive band specific to sex. Then the primers were tested in RNA extractions of the larvae, which can not be easily differentiated sexully by their morphology and the resulting amplification of RT-PCR shown in the image of the gel (B). The larvae show a clear band pattern in which males of females are unequivocally distinguished. The gel image (C) shows a band approximately 600 bp of RT-PCR using aedessxFI and aedesxR2 (SEQ ID No. 66) of the individual male and female pupae primers. The sequence of this band showed a specific female variant of the junction which does not seem to possess the male shared exon to which it is predicted that aedesxR5 will join (exon 7, see figure 56). Molecular weight markers are shown in the kb (Eurogentec SmartLadder ™, the sizes are approximate). Figure 47: Diagrammatic representation of the dsx gene of Stegomyia aegypti (non-scalar). A fragment of the dsx gene of Stegomyia aegypti is depicted above. Exons 5a and 5b are female specific and exon 6 is a male specific exon. Two splice variants specific to females have been found (F1 and F2) that span exons 1-4, 5b, 6 and 7 (F1) or I-4, 5a (F2); transcripts in males (M1) encompass exons 1-4, 6 and 7 but not exon 5a or 5b and a transcript (C1) of 1-4 and 7 but not exons 5a, 5b or 6 are shown in males and females. The numbers for each of the exons after # refers to contig 1.370 (http://www.broad.mit.edu/annotation/disease vector / aedes aeqypti /), which is read in the opposite orientation, and after * refers to the nucleotide sequence shown in SEQ ID No. 43. Figure 48: Diagrammatic representation of the dsx gene of Stegomyia aegypti.

The entire dsx gene of Stegomyia aegypti that is represented on exon 5 is the female specific exon and exon 6 is an assigned male specific exon. The principle, transcripts in females encompasses exons 1.2.3.4.5 and 7, and those in males encompass exons 1.2.3.4.6 and 7. The numbers for each of the exons after # refers to contig 1.370 ( http://www.broad.mit.edu/annotation/disease/readura vector / aedes aeqypti /) which is read in the opposite orientation, and after * refers to figure 12. Figure 49: Map of pLA plasmid 1172. A region of the tTAV coding has been put under control of a fragment of the actin-4 gene of Stegomyia aegypti (Muñoz et al. 2005) which includes the 5 'UTR, first intron, and upstream sequences (assigned promoter). The construction also contains an Insect tet07 sequence. The construct has piggyBac ends and a DsRed2 marker for stable integration into a genome. Figure 50: Sex-specific junction of tTAV in LA1172 transformants. The image of the RT-PCR gel of RNA extracted from line 2 of male and female pupae of LA 1172. The primers used were Agexonl (SEQ ID No. 67) and Tra (tTAV) seq + (SEQ ID No. 68). The sequence of the RT-PCR bands showed the expected splice occurred in males and females. The data shown in the previous diagram are for line 2, line 8 of LAI 172 showed exactly the same results (data not shown). The markers are SmartLadder ™ by Eurogentec; approximate sizes are indicated, in kb). Figure 51: RT-PCR of native type samples, showing the sex-specific variants of the Actin-4 gene splicing of Stegomyia aegypti. Image of the RT-PCR gel of RNA extracted from various stages of development, and dissections of adults, of line 8 of LAI 172. The primers used were Agexonl (SEQ ID No. 69) and exon 3 (SEQ ID No. 70). The gel image shows that strong expression of the Actin-4 gene occurs only in the chrysalis stage, and that adult expression is generally limited to the female thorax where the flight muscles are located. Table 17, below shows the content of each lane.

E = accumulation of ~ 100 embryos MH = head of adult male L4 = 4a larva instar MT = adult male thorax ME = young male chrysalis (<4 hours old) MA = male adult abdomen FE = young female chrysalis (< 4 hours old) FH = adult female head MP = male chrysalis FT = adult female thorax FP = female pupae FA = adult female abdomen -ve = control of the tablet Table 1. Other examples Example 1: moths We have reconstructed the constructions based on our transient expression data using a recombinant construction of the migen derived from Bom byx mori. This is described in more detail below in the section entitled "Alignment of the dsx sequence of the polymer and the conserved motifs" Example 1 1: Use of Bztra We have done two constructions based on Bztra again, expressed in Mex fly (LA3376) . LA3376 produces a specific repressible lethality to females. LA3376 that we have previously shown to work and correct correctly in the Mediterranean fly. Transformants in the fly Mex. { Anastrepha ludens) were also generated with LA3376. These were analyzed for the correct splicing of the Bztra intron to demonstrate the phylogenetic range of the Bztra intron by RT-PCR using the primers SRY and AV3F (Figure 1 5 and "Gels for RT-PCR for the fly Mediterranean ", previous section). This shows the correct palming of the Bztra intron in the Mex fly.

Example 12: Dmdsx in the Mediterranean fly (DmDsx in the transgenic mediteraneo fly example: insect fusion in # 797) We have also again collected data in a Dmdsx construction in the Mediterranean fly. The construct used a doublesex gene fragment from Drosophila melanogaster to give the sex-specific expression of a fragment of the NippIDm gene from Drosophila melanogaster (we call this fragment; "insect "). We did not see a clear connection specific to sex. However, the phenotypic data demonstrate a certain specificity to sex; We saw the increasing lethality of females, penetration of approximately 75%. Of course this incomplete penetration could be due to the level of expression, the lack of insect toxicity in the Mediterranean fly, etc. We also had a significant reduction in the number of males, but the tTA source, LA670, used in this experiment could by itself kill some of the males. We have tested three independent transgenic lines of the meditarraneo fly that take a fusion of the insect to the sequence of DmDsx that was thought to be expressed specifically in females. This construction may not have worked perfectly, possibly due to the essential sequence for the alternative correct splice and / or the Sxl link points required by DmDsx, and since the fly of the Mediterranean does not use Sxl in the determinant form of sex, DmDsx may not be able to fully buttress this fusion in the correct way in the Mediterranean fly. However, we succeeded in reproducibly causing an increasing lethality in the females compared to the males through the three lines with a very similar efficacy (approximately 75% of greater lethality observed in females than in males). This demonstrates the dsx system can work through distantly related species (evolutionary separation is around 120-150 million years ago), and if the Ccdsx sequence was used it may have worked well due to the Sxl requirement of Dmdsx. The 797 results are shown below, using a splice insect system dsx Tet014 (Pub EGFP). They show that this system is lethal in the larval stage (- 50%), and it is likely to be successful in females (- 75%). 797 is marked with green (G), 670 with red (R) .670 is a source of tTAV, so one expects to see a phenotype in R + G flies; G (and R) are only controls. NF - non-fluorescent (ie, native type) is also a control where it is included. All the progeny were raised in free tet media. The three independent lines seem to act in a similar way. 797A797A M2 x 670A / +: Adult Chrysalis Machos: Hembr a s G 184 176 85: 91 R + G 74 57 44 13 77C / 797C MI x 670A / +: Adult Pupa Machos: Hembr a s G 169 157 89: 68 R + G 94 67 54: 13 797G797C M2 x 670A / +; Cris alida s Adult s Ma cho: Hembr a s O 406 377 179: 1 8 R + G 171 147 121: 26 670A / + 797C / + M2: Pupaults Adults Machos: Females NF 198 192 92: 100 G 162 147 67: 80 R 149 72 43:29 R + G 45 22 20: 2 Average of the 3 lines: number of females of R + G = 21% of the number of males of R + G, therefore excess of substantial lethality in the females in relation to the males of R + G. This effect is not observed in the control females of R alone or of G alone, nor in the native type. Examples 13-15: We have demonstrated again: (5) sex-specific splicing in minigen constructions based on recombinant Aadsx (6) sex-specific phenotype of a construction based on Cetra; and (7) sex-specific splicing in constructions based on Actin4 from Aedes. At least some of each of these examples not only shows minigenes, but actually shows the splice to generate tTAV / tTAV2 or ubi-tTAV2 Example 13: Aedes doublesex minigenes (dsx) See also the section titled Link points to Tra2 of Aedes dsx. We have isolated the dsx gene of Aedes aegypti (Aadsx) and identified 6 transcripts of this region (figure 1): These are: 2 transcripts specific to the male (mi and M2), 3 transcripts specific to the females (F1, F2 and F3) and a transcript found in males and females (intermediate frequency). We made two constructions of the minigen. In these constructions, it suppressed the great majority of the intronic sequences. For example, DSX minigen 1 is approximately 4.4 kb in length, while its terminal sequences are separated by approximately 26 kb in their natural context, ie in the genomic DNA of Aedes aegypti. The splice in the minigen2 of Figure 1 is illustrative as the splice occurs in the "female" form in males and females. This may mean that this system depends on the alternative use of the splice acceptor. In this model, there is competition among the alternative acceptors of the joint, with a certain factor specific to sex predisposing this, the factor specific to sex It's probably Tra. But suppressing the splice acceptors M1 and M23 'forces the splice into the F forms, by eliminating the alternative. Therefore, it is preferred that one or more female-specific 3 'enripíame acceptors (F1 and / or F2) be provided along with an additional 3' splice acceptor. More preferably, the additional acceptor of the splice is the splice acceptor 3 'of the splice variant M1 or M2 (or both), although it is considered that this is not essential as it is known that other known splice acceptors 3'; It is likely that they work. Figure 1 illustrates the various transcripts produced by the alternative splicing of the doublesex gene from Aedes aegypti (Aadsx). It will be appreciated that Aedes aegypti is also known as Stegomyia aegypti. The figure shows the Aadsx gene of the fourth exon, which is not alternatively spliced, that is, it is present in all the transcriptions discussed here. The enumeration starts from the first nucleotide of the fourth exon (acgacgaact ...). Note that the diagram is not to scale - the introns are much longer than the exons. The alternatively spliced region in total covers more than 43kb. This fragment of the minigene was included in an expression construct (LA3515). Transgenic Aedes aegypti was generated by site-specific recombination at an attP site, using the method of Nimmo et al. (2006: Nimmo, D.D., Alphey, L.

Meredith, J.M. and Eggleston, P (2006). High efficiency site-specific engineering of the mosquito genome. Insect Molecular Biology, 15: 129-136) A second smaller minigene was constructed in a similar way (minigen2 DSX) and an expression construct for this was inserted in the same atfP site as the DSX minigen 1, to allow direct comparison (LA3534). Minigen 2 did not show sex-specific splicing. This indicates that the sequences present in the DSX minigen 1 but not in the DSX minigen 2 (approximate 2029bp, see FIG. 1 and SEQ ID No. 150, where the exons are in positions 29-163 and 1535-2572 ) are essential for the correct alternative splice, although the first intron spliced alternately, and the exonic sequence that flanked it immediately, are present in both constructions. We have produced two transgenic lines (LA3491 and LA3534) using minigens constructs of the Aedes aegypti dsx gene. LA3491 is a fusion of the shared exon4, the exons-specific to the females of the cartridge, and part of the first exon 3 'shared; (exon 5 in the M1 transcript). The transcripts of the LA3491 minigene region were analyzed by reverse transcriptase PCR (RT-PCR) and sequence formation. The transcripts that corresponded to the alternative splice in the form F2 were found in females but not in males (figures 2 and 3) and in the form F1 had a certain masculine expression but very low (figure 4). While the transcripts that corresponded to the M1 form were detected in males but not in females (figure 2). Since the minigene did not contain the 3 'splice acceptor of the M2 variant, this transcription was not possible with this construction. This minigene does not contain any exogenous sequence, although it clearly demonstrates the sex-specific splice of an Aadsx fragment, in fact a fragment of highly suppressed "minigene". It will be apparent that certain sequences are important for controlling the splice and should therefore be retained, as discussed elsewhere. This can be easily established by the deletion of certain portions and the alternative splice test by RT-PCR for example. Figure 2 shows RT-PCR of males and females of the transgenic line of aedes aegypti LA3491 using primers 688 - iel transcr (SEQ ID No.4) and 790 - Aedsx - m - r2 (SEQ ID No.5). Using those primers, the splice in the F2 pattern would give a band of approximately 985bp while the splice in the M1 pattern would give a band of approximately 516bp. A band of approximately 985bp (F2) appeared only in lanes representing the females and a band of male-specific transcript 1 of approximately 516bp appeared only in the males. These bands have been sequenced and show that the correct splice had occurred, ie the type F2 and type M1 respectively. The absence of bands in the controls without RT (-RTCON) shows that there was no genomic DNA contamination in the samples. Lanes 1 and 11 are markers (SmartLadder ™ of Eurogentec, bands of 1.5kb to 0.2kb are indicated). Lanes 2 and 3 are negative controls (without reverse transcriptase) and lanes 2-9 represent the reactions performed in the extracts of males or females as marked. Figure 3 shows RT-PCR of males and females of transgenic LA3491 lines of Aedes aegypti using the 688-iel-transcr primer (SEQ ID No.4) and 761 - the Aedsx-fem-r (SEQ ID No.6). Using these primers, the splice in the F2 pattern would give a band of approximately 525bp. A 525bp band was approximately present in reactions in female extracts, but not corresponding reactions in male extracts. The sequence of this band of 525bp confirmed that it was correct, that is to say that the splice of type F2 had occurred. Marker (Eurogentec SmartLadder ™, bands from 1.5kb to 0.2kb are indicated). Figure 4 shows RT-PCR of males and females of transgenic LA3491 lines of aedes aegypti using primers 688 - iel-transcr (SEQ ID No. 4) and AedsxRI (SEQ ID No. 4). Using these primers the splice in the F1 pattern would give a band of 283bp. A band of 283bp is present predominantly in females, although there is evidence of a small amount of splicing in males. The sequence confirmed that this band actually corresponded to the splice in the F1 pattern. Marker (Eurogentec SmartLadder ™, bands from 1.5kb to 0.2kb are indicated). LA3534 is identical to LA3491 except for a 3 'deletion of approximately 2kb. This construction did not show any differential junction between males and females (figure 1, minige 2). The RT-PCR gels were not shown for this case. According to these results, several constructions have been designed to incorporate sex-specific splices of LA3491 (Figure 1, minigen 1) into a positive feedback system. LA3612 (Figure 5), which incorporates a fusion of ubiquitin and tTAV2 in the region of the dsx coding, is designed for when female F2 transcription occurs, ubiquitin breaks down and tTAV2 is released to initiate and sustain the system. positive feedback LA3619 (figure 5) has tTAV2 without ubiquitin and uses its own start codon of translation. LA3646 (figure 5) is identical to LA3619 except that the start codons for the dsx gene have been mutated; this should improve the amount of tTAV2 produced by removing the non-specific translation. Figure 5 is a diagrammatic representation of the plasmids based around the splice in the dsx minige of Aedes aegypti. For greater clarity it will be understood that the first female intron represents any of the F1, F2 or F3 junction, and the tTAV in the diagram refers to tTAV2 (it will be appreciated that other proteins or other versions of tTA or tTAV could be used alternatively). In each of these plasmids, apart from LA3491, the heterologous sequence has been added to exon F2. "ATG assigned"; represents any sequence of the 5 'ATG trio located in the exon sequence relative to the heterologous DNA. In LA3646 these assigned start codons of the translation ("assigned ATG") were removed or modified. In the case of the LA3612 construct, the translation of upstream (5 ') ATG that is in frame with the coding region of the ubi-tTAV will still produce the functional tTAV (assuming no stop codon intervenes), after the separation of the halves of ubiquitin and tTAV by the action of the protease. The various alternative splicing cartridges are operatively linked to a convenient promoter, a transcriptional adapter and other regulatory sequences. This example demonstrates the sex-specific splicing of a highly compressed fragment of a "minigene" in a heterologous context (ie a heterologous promoter, 5 'UTR and 3' UTR). Although they do not show the differentiated expression of a non-Aedes sequence, as the alternatively spliced exons are derived from the Aadsx gene and do not contain additional material, it clearly illustrates the viability of this modality. In any case, the promoter 5 'UTR and the 3' UTR are heterologous. We have additional constructions that illustrate several different methods to obtain the expression (sex specific) differentiated from a heterologous protein by this dsx. Alignment of the sequence of TRA Pane et al. (2002) suggested that certain sequences that are related to the known binding sites of the Tra / Tra-2 complex in Drosophila could be important in the regulation of the Cetra junction, and this is also known for Drosophila dsx and has also been suggested for the dsx of the Anopheles gambiae (Scali et al. 2005). The consensus sequence is variously described as UC (U / A) (U / A) C (A / G) AUCAACA (Pane et al.), SEQ ID NO. 8, or UC (U / A) (U / A) CAAUCAACA (Scali and others 2005), SEQ ID NO. 9. It is important that these definitions are extremely similar. Pane and others identify 8 partial matches to this consensus in the sequence of Cetra (7 or more nucleotides that co-exist with the consensus sequence of 13 nucleotides, Scali and others identify 6 co-angles in Agdsx (9/13 or better). they are known to regulate the alternative splicing of the fruitless gene (sterile) of Drosophila, Scali and others review 3 matches in that sequence (12/13 or better) The correct splicing of dsx may also require a rich purine region, as discussed Scali et al. As can be seen in Table 2 and Figure 7, we have identified what are probably the significant cleavages of binding sites for Tra / Tra2 in the mix of Aedes aegypti dsx.

Alignment of the moth dsx sequence and conserved motifs Figure 6 shows an alignment of the second exons -specific to the females and of the flanking sequences of the dsx genes of the pink grusano (Pectinophora gossypiella, PBW-dsx, SEQ ID No. 146 ), the silkworm (Bombyx nori, bombyx-dsx, SEQ ID No. 147) and the apple moth (Cydia pomonella, apple dsx, SEQ ID No. 148). The second exon specific to the sample is bold. We identify multiple copies of a repeated short nucleotide sequence, conserved in sequence and of an approximate location between these related moths in a relatively distant manner; these are located 5 'from the female specific exon. The conserved repetitions AGTGAC / T are underlined. The asterisks (*) represent the identical nucleotides, the dashes (-) represent the spaces for the best alignment. The exons are represented in SEQ ID No. by the following numeration of the nucleotide: SEQ ID NO. 146289-439; SEQ ID NO. 147 339-492; e SEQ ID No. 148285-439. Link sites of Aedes dsx Tra2 In females of Drosophila melanogaster, Tra and a product of the active constitutive gene tra2, they act as splicing regulators when binding to the splice booster sites in the dsx pre-mRNA, which activates the site of the weak 3 'acceptor; of the specific exon to females (Scali and others). In males there is no TRA expression and the weak 3 'acceptor site is not recognized and the splicing occurs in the male exon. To search for Tra / Tra2 assigned binding sites we used the consensus sequence of these linkage points deduced for Tra / Tra2 from Drosophila and we looked for the distribution of these in the sequence of the dsx gene of Aedes aegypti. This is shown in table 2, which follows. 33 acatcgattcaca 16085 10 6 [H5_ 34 cgctca tcaaca 16175 10 5 116 tctaccataaaaa 16511 10 5 117 36 aaa gaatcaaca 20044 10 5 118 37 acatcgttcaacg 21374 10 5 119 38 tcttgatitcacca 215S0 10 < 5 120 39 tctgcagacaca 22408 10 < 5 121 L O tcttcggtaatca 23285 10 5 122 41 1 tctataaaea & SI 25436 10 < 5 123 42 1 taaacaal-.fí.¾ata SI 25440 10 6 124 43 'taaacaagc.aaaa' 28242 10 5 125 44 tcaacga cggcg 30309 10 6 126 45 tgatccatcatca 30910 10 5 127 46 -. 46 -Aacatgcaaga 32295 10 < 5 128 47 i. taaabaeaga 32862 10 5 129 48 tcaaagatctata 40551 10 5 j 130 49 Laatgaattaaca 40847 10 5: 131 50 tttaccatcaaci 41712 110 5! 132 1 taatgaaaeaaca 43380 10 5 133 52 * gtttcaattaaaa SI 13500 9 6 134 53 * tattcaat ataa SI 13602 9 6 135 54 * tcttcaatecttt I 15002 9 6 136 55 * tcaacgatccttt SI 15533 9 6 137 Table 2: * = in 3491, only 9/13 but 6/6 in the core. This table does not include 9/13 identities in addition to those in 3491 with 6/6 identity with the core sequence of wwcrat. This core consensus sequence (WWCRAT) is particularly preferred. Figure 7 is a diagrammatic representation of the binding sites assigned Tra / Tra2 within the coding region of plasmid LA3491. This diagram is approximately scaled and represents a sequence of approximately 4kb. We can calculate the chance of a random match to the Tra / Tra2 consensus sequence. Assuming that all 4 nucleotides occur with an equal frequency, the probabilities that any given nucleotide in a random sequence is the first nucleotide of a 10/13 match or better than the consensus is approximately 7XIO "4. Therefore, one would expect slightly less than one match of that type for every 1000 nucleotides of such random sequence The calculation for this is below Sex-specific splicing: probabilities Questions A consensus sequence of the binding point consists of 13 bases Ten of those (fixed) positions (let's call this system X) must each one being a specific basis, the other three (let's call this system Y) can each be one of two specific bases, assuming that each possible base A, G, C and T is equally probable and that the base in each position is independent of the bases in the other positions, what is the probability that a sequence of 13 bases randomly selected coincides exactly with this sequence? What are the probabilities of such a sequence being an almost a mismatch (which allows up to one, two, three or four differences)? The answers are given in Table 2 below and the procedures are shown after that table.

Answers Table 3 Procedure P (exact collocation) - Pa ~ = 1.192x10"' at 3 d.p. (3 d.p. everyone who follows) P (mismatch in exactly 1 position) = P (mismatch in one of the 10 X positions or mismatch in one of the 3 Y positions) = ./ > = 10] riYrsYiV +, 3 jiYVit .oo * 3) +3 33 3.93 x10"U) U? 2 J 419 x 23 P (mismatch in exactly 2 positions) = P (mismatch in 2 of the 10 positions) X or mismatch in one of the one of 10 positions X and 1 of the 3 positions Y or no coincidence in 2 of the 3 positions Y) + I0x iV sYiV iWtY 2J 2) ((45 x 3) - (30? 3) + 3) _ 498 _ 249 _,? _, ^ 23 2. "' P (mismatch in exactly 3 positions) = P (mismatch in 3 of the 10 positions X or mismatch in 2 of the one of 10 positions X and 1 of the 3 positions Y or mismatch in 1 of the 10 positions X and 2 of the 3 positions Y or no matches in the 3 positions Y) (120 33) + (45 33) + (30x) + l) = 5356 _ 1339 = g5? , = * 2"" 2a * "~ 2" ~ P (mismatch in exactly 4 positions) = P (mismatch in 4 of the 10 positions X or mismatch in 3 of the one of 10 positions X and 1 of the 3 positions Y or no coincidence in 2 of the 10 positions X and 2 of the 3 positions Y or no matches in 1 of the 10 positions x and 3 of the 3 positions Y) P (mismatch in up to 1 position) 1 + 33 17, 23 * 22 «4.053x10" P (mismatch in up to 2 positions) "1 + 33 + 498 532 133" i¾ + Pi + P2 ^ G- - ^ r = ^ r = 6J42xlO-s P (no match in up to 3 positions) 1 + 33 + 498 + 5356 5888 = + > ] ÷ 2 + P3 = P (no match in up to 4 positions) 1 + 33 + 498 + 5356 + 27975 _ 33863 -P + P1 + P3 + ¡+ = - 4.037x10, - ·? Experiment 14: Cetra We have a line of LA3097 (LA3097A) that shows the very good expression of its fluorescent marker; it is unknown if this line is a single integration event. This line shows evidence of sex-specific splicing, when raised without tetracycline all females die as embryos, and when they are under 30 g / ml of tetracycline, males and females survive. This example is important. It shows that Cetra provides an alternative platform specific to sex in aedes, and that this can be used to give sex-specific lethality. This, therefore, provides evidence of the phylogenetic range for the Cetra junction. Thus, it is quite plausible that the present invention can be applied to all diptera, since we have shown that Cetra functions in Drosophila, tefritides and mosquitoes, which essentially extends to the entire order of Diptera. It is surprising that Cetra works in the aedes, given the rapid evolution of the tra sequence. We transform Aedes aegypti with the construction LA3097.

The heterozygous males of the resulting transgenic line were crossed with those of the native type and the progeny were bred in aqueous medium supplemented with tetracycline at a final concentration of 30 pg / ml. Adults were recovered in the following way: 14 males and one female, thus demonstrating a significant specific lethality to the females. These species and strain normally have a sex ratio of approximately 1: 1, therefore this construction gave specific female lethality in Aedes aegypti. The equivalent constructions that did not contain the intronic sequence of Cetra had non-specific lethality to sex. Therefore, the Cetra intron can be used to provide differential regulation (this is sex-specific) of gene expression in mosquitoes, and this can also be used to provide sex-specific lethality and a method for the selective elimination of females of a population. In more detail: in 0 pg / ml of tetracycline, males survive only until they are pupae, that is, they do not reach adults. The females die so early that we do not see them, probably as embryos, so it's still a Differentiated effect between the sexes. However, the pupal lethality in males suggests that the system is not completely off in males. The only line of insertion that we recover is unusual, because it demonstrates the extremely strong expression of the marker; other insertions with more typical levels of the expression could not demonstrate the male lethality well. Splicing in LA3097A LA3097 splicing analysis of transgenic mosquitoes LA3097A by RT-PCR demonstrated that males and females shared two transcripts, a band of approximately 950 base pairs and a band with less bp approximately 800 (figure 59). The sequence of these bands showed that the band with -900 bp corresponds to a non-specific variant to the sex of the splice (AeM2, bp -920), and the weakest band was a mixture of a non-specific variant to the sex of the splice (AeMI). , ~ 804bp) and the female form (AeFI, ~ 765bp), see figure 60. The splice of the AeFI transcript was identical to that shown for this construction in the Mediterranean fly (figure 33). The splicing of the transcripts of M differs somewhat considered in the native context (Cetra that is spliced in the Mediterranean fly, or the native gene or as we observed from LA3097 in the transgenic Mediterranean fly); in AeMI the second exon alternatively spliced (MEIb) is not included in the mature transcription of AeMI and in AeM2 the second alternatively spliced exon (ME2b) is not included equally in the transcription matures AeM2. In other words, the first but not the second exon of the cartridge, relative to the prototype of the Mediterranean fly, is present for each of these transcripts. Note that as a consequence of the absence of the second exon of the cartridge in AeMI, and the reading frame of tTAV2 relative to the first exon of the cartridge in this construction, the splicing in the AeMI pattern does not lead to the interruption of the open reading frame tTAV2 , but rather to the addition of 39 nucleotides (corresponding to 13 amino acids) between the ATG and the rest of the tTAV2 of the open reading frame. It is likely that this variant of tTAV 2 retains some activity, related to normal or prototypic tTAV2 (as encoded by the F1 splice variant). In the absence of tetracycline, a phenotypic effect was observed in males as well as in females, although weaker in males than in females. The production of a partially active variant of tTAV2 from the transcription of AeMI in males (and females) may explain this. Figure 59 - shows RT-PCR in males and females of the transgenic aedes aegypti line LA3097 using the primers HSP (SEQ ID No. 139) and VP16 (SEQ ID No. 140). Using these primers, the splicing in the CcFI pattern (ie corresponding to the F1 variant of Ceratitis capitata) would give a band of approximately 765bp and splice in CcMI 1005bp and CcM2 1094bp. In males and females, a band was observed strong of about 950bp (1) together with a weaker band of about 800bp (2). Marker (Eurogentec SmartLadder ™, bands from 1.5kb to 0.4kb are indicated). Sequence analysis of several clones from band 2 (ie splice variants of AeMI / AeFI) of males and females showed that one of five female clones showed splicing of AeM2 (20%), while in males three of all four copies showed splicing of AeM2 (75%); the rest of the copies showed AeFI splicing. This indicates that there is more transcription of AeFI present in females than in males and this would explain the differentiated effect of the killing observed among them. Figure 60 illustrates the various transcripts produced by the alternative Cetra splicing of the Aedes aegypti transgenic line LA3Q91A. 3097 represents the sequence of the Cetra DNA and the numbers relate to the figure described elsewhere. Shading and boxes are also related to figure 33. Note that the diagram is not to scale. Example 15: Actin-4 of Aedes; We have eleven lines of LA3545, which uses the actin-4 gene of Aedes (AeAct-4 or AaAct4) to lead to the expression of tTAV2. In construct LA3545, a sequence encoding tTAV2 has been inserted into the second exon of AaAct4 (FIG. 10). For transcripts spliced in the characteristic pattern of AaAct4 spliced into females, the ATG of the coding region tTAV2 will be the first (mostly 5 ') ATG of the transcript. The splicing in the characteristic pattern of AaAct4 that is spliced in males introduces an array of start and stop codons before the tTAV2 sequence that tends to inhibit or interfere with the translation of the ATG of the tTAV2 coding region. These lines should express only tTAV2 in female pupae. The splice is shown in figure 8, ahead. Figure 8 shows RT-PCR of the male and female adults of the transgenic Aedes aegypti line of LA3545AeC using the primers AgexonlF (SEQ ID No. 141) and TETRRI (SEQ ID No. 142). Using these primers, splicing in a pattern equivalent to that of the native AaAct4 gene would give bands of approximately 347bp for the female-type splice variant and approximate 595bp for the male-type splice variant. A band 347bp of the approximate band (F) was found only in reactions in the extracts of females; a band of approximately 595bp (M) was found in males and females. The sequence has confirmed that the correct splice occurred in males and females. Marker (Eurogentec SmartLadder ™, bands from 1.5kb to 0.2kb are indicated). We also have transgenic aedes aegypti LA3604, which is similar to LA3545 unless it has a directed start codon in the exon 1 portion that is present in male-type and female-type transcripts (figure 10). This one is arranged to be the first ATG in any type of transcription. LA3604 encodes tTAV2 fused to ubiquitin (LA3545 codes to tTAV, while LA3604 codes to ubi-tTAV2). This construct should produce a fully functional tTAV2 protein in females only, even if the male form is expressed in females the additional male exon contains several of the start and stop codons that would prevent the translation of the Ubi-tTAV2 fusion protein. The alternative splicing of AaAct4 occurs in the 5 'UTR (of the native gene). It may or may not have a regulatory role in the native gene. One possibility is the following: in the female-specific splice variant, the start codon of the AaAct4 coding region is the first ATG of the transcript. However, in the male-specific splice variant there are several additional 5 'ATG sequences; to the start codon of the AaAct4 coding region; most of these have stop codons in the frame at a short distance 3 '. This arrangement of the sequence can interfere with the efficient translation of the AoAct4 protein and thereby reduce the expression of the protein in males with respect to females. This is the arrangement in LA3545. However, a greater differentiated effect between males and females would be expected if the intron was included in the coding region (more than 5 'UTR), ie inserted between the start and stop codons of the polynucleotide for expression in the body. In this case, the exon specific to the male of The cartridge would change the potential of the encoding of the transcript, and not just by simply interfering with translation.

This is achieved in the construction LA3604. We modified the first shared exon to include an ATG sequence in a convenient context of the sequence for translation initiation. In this modified sequence, this is the first ATG in the male (M) or female type (F) type splice variants. After splicing in the F form, this 5 'ATG (modified) is in frame with the coding region of ubi-tTAV. Type F transcripts would therefore encode a fusion protein, encompassing the sections encoded by (i) the part of what is normally Act4 5 'UTR (but here obviously translated, and thus nothing of UTR at all), (ii) ubiquitin coding region and (iii) coding region of tTAV2. The activity of ubiquitin cellular proteases will produce the tTAV2 protein. The translation of the modified 5 'ATG would terminate by stop codons in the frame in the additional sequence (exon of the cartridge) presnete in transcripts spliced in the form of M. This would therefore prevent the expression of functional tTAV2 in males, thus giving the sex-specific expression of tTAV2. Obviously, this gives a general method for the sex-specific expression of a protein, substituting the tTAV2 segment for another protein or the sequence of interest. Using this strategy we have provided transgenics and we have demonstrated the specific connection to sex (figure 9). Figure 9 shows RT-PCR of males and females of the transgenic line LA3604AeA of Aedes aegypti using the primers AgexonlF (SEQ IDNo.141) and TETRRI (SEQ IDNo.142). Using those primers, the splice in the female form would give a band of approximately 575 bp, whereas the inclusion of the specific exon to the male of the cartridge would increase this to approximately 823 bp. A band of approximately 575bp was observed in each analyzed female, while a band of approximately 823bp was observed in each male analyzed. These bands appear to be substantially specific to the respective sexes. The sequence of these bands showed that the correct splice had occurred in males and females. Marker: Eurogentec SmartLadder ™, bands from 1.5kb to 0.2kb are indicated. Figure 10, below, is a diagrammatic representation of plasmids LA3545 and LA3604. S1: shared exon 1; MI: additional sequence included in the male-specific exon 1; S2: shared exon 2 (only 5 'end); ubi: ubiquitin coding sequence; tTAV2: coding sequence of tTAV2. In several of the LA3545 transgenic lines, a specific effect on sex and tissue was observed: females can not fly. Two of the lines show a female phenotype that can not fly 90-100%, one line shows 70% lack of flight and the other 50%. This phenotype is probably due to the expression specific to tTAV2 females in developing flight muscles. The difference in phenotypes between the lines is due to the positional effects on the expression of the AaAct4 promoter. Because the position of the genes in the expression of the genome can be influenced by a number of factors (the heterochromatin or euchromatin regions, the reinforcing and suppressor elements, proximity to other genes) that can be easily observed in the fluorescent markers used to identify transgenics. All eleven LA3545 lines were identified because they have different fluorescent profiles, although they have the same promoters and markers. This variation is due to positional effects. This would then mean that we would expect some LA3545 lines to express more tTAV2 than others due to positional effects, and those lines that express more would produce a specific flight failure phenotype for females.

To test this hypothesis, we developed a separate line of Aedes aegypti with a tetO-DsRed2 reporter gene (LA3576 see SEQ IDNo.137 from figure 17and), when crossed with the various lines LA3545 would allow the visualization of where and when Actin4- is expressed. tTAV2. Of the 8 LA3545 lines crossed to LA3576 all showed indirect fluorescence specific to flight muscle females in late L4 larvae, pupae and adults. In four of the lines the expression of DsRed2 appeared to be specific (ie exclusive) to the female indirect flying muscles; in the other four additional tissues it showed the expression of DsRed2. This phenomenon, in which the expression of a transgene depends in part on the region or point in the genome into which it has been inserted, is called the position effect, and is well known and understood by the person skilled in the art. Using LA3576 it was proved that the expression of tTAV2 in LA3604 was specific to females, occurs mainly in the indirect muscles of flight and is specific to the stage. Several different constructions of the tetO effector were then constructed to analyze their effects. The transgenic tetO-MichelobX (LA3582, see figure 15 and SEQ ID No. 144) when crossing it with LA3545 all showed phenotypes with specific flight failure to the females could be repressed by means of tetracycline. This proves that Actin4 can be used to drive an effector gene in a manner specific to stage, tissue and sex. Because some lines of LA3545 had a specific phenotype to females without the presence of a determinant-induced gene, it was shown that tTAV2 could act as a determinant molecule. tTAV2 is composed of a tTA, a mandatory domain and VP 16, a tetO protein of the herpes simplex virus. VP 16 activates the transcription of immediate early viral genes using their amino-terminal sequences to be enriched to one or more proteins encoded by the host that recognize DNA sequences in their promoters. In LA3604 a tetO-VP16 determinant gene has been added to enhance the effect of tTAV2. In three transgenic lines of LA360 this has led to a 100% of the specific vallet flight phenotype to females when they are reared without tetracycline, demonstrating that VP 16 is an effective effector molecule. Note that LA3604 has a modified potential start codon 5 '(ATG); the intron alternately spliced. Therefore, in this construction, it is expected that the male-specific exon will interrupt the tTAV of the coding of the open reading frame (ubi-tTAV); since the sequence specific to males contains several stop codons, this will tend to reduce or eliminate the production of functional tTAV in males. By comparison, the male-specific exon is 5 'to the start codon of tTAV in LA3545. However, by inserting a number of start codons 5 'to the start codon of the tTAV gene (which is the first ATG of the female transcript but not of the male transcript), none of these additional start codons are suitable for production functional tTAV efficient because they are out of frame or because they have stop intervention codons, this arrangement will also tend to reduce or eliminate the production of functional tTAV in males, consistent with previous phenotypic data. Example 16: use of ubiquitin and intron placement We have again made Cetra-based constructions with the Cetra intron cartridge in a variety of different contexts, ie flanked by different sequences. The various transgenic Mediterranean fly lines that carry them have been built. This shows that the system is general and robust, ie it will work for a wide range of heterologous sequences of interest. We also have at least one newly performed example of a fusion of Cctra-ubi-tTAV that gives the correct splice (DsRed-cctra-ubi-tTAV). Preferred examples of the functional protein place the coding sequence either for ubiquitin or tTA, or their functional mutants and or variants such as tTAV, tTAV2 or tTAV3, 3 'to the intron. They are arranged so that these elements are substantially adjacent to the 3 'end of the intron, preferably in such a way that the coding region begins within 20 nucleotides or less of the 3' intron limit), and more preferably, immediately adjacent to the 3 '. end of the intron, although this is less relevant if the Ubiquitin system is used. Preferred examples of constructions according to the present invention are listed in Figure 4, below. It will be appreciated that LAI 188 is not within the scope of the current invention, as it does not encode a functional protein, ie it does not function correctly. This is probably due to the unexpected use of 4 bp 5 'emplame donor at the junction with the Cetra intron sequence, leading to a' frame shift 'mutagen that is induced at all splices. Therefore, it includes for informational reasons only.

Table 4 Figure 4 shows the constructs containing a splicing control sequence that is derived from an intron tra. The introns were derived from C capitata (Mediterranean fly), C. zonata or B. rosa (see column 2). The intron was inserted into the coding region such that the distance between the assigned ATG primer and the last nucleotide of the exon immediately anterior to the intron as it should be indicated in column 3. The intron is inserted into or adjacent to the region for either ubiquitin, tTAV, reaperKR, nipper or ubiquitin- DsRed according to the indications in column 4. These were generated and shown to splice successfully, by RT-PCR or phenotypically in the Mediterranean fly and, in some cases, also in Drosophila melanogaster (LA3077) or Anastrepha ludens ( LA3097, LA3233, LA3376). In addition, the distance between the ATG and the end of the exon immediately before the Itron intron (assuming the splice type F1) can be extended from Obp to at least + 949bp without adverse consequences for the splice (see Table 4)., column 3). Thus, it is reasonable to assume that this distance can be up to at least 900 bp and preferably up to at least 949 bp. Additional information on these examples is summarized in Figure 5. The preferred option is to not use any endogenous sequence to achieve the correct alternative splicing control of the expression (+ 0bp in Figure 4). We prefer to insert the intron between the TG ... GT flanking dinucleotides in the coding region of the protein of interest to be alternately spliced to ensure correct splicing since this may be important, however we will not restrict ourselves to this if necessary since other flanking nucleotides can also function correctly. Examples of LA1038, LA3054 and LA3056 they include a certain exogenous endogenous flanking sequence of the natural Cetra gene. In Table 5, if 6 nucleotides or less are included (start codon including ATG) of particular mergers at 3 'or 5' of the junction junction, for the purpose of summarizing in this table these will not be considered part of the fusion. Table 4 can be correlated with Figure 3 to find which intron (Cetra, Bztra or Crtra) is used in each example. Once again LA1188 is included for informational purposes only and does not fall within the scope of the present invention.

No. of the intron is, the intron is sequencing tra exoiuca sequence tra exonica construction fused to 5 * fused to 5"(bp) fused to 3" (bp) (Figs # 0 fused to 3"LA 1188 (80) Hsp70-tTAV cTAV + 0bp + 0bp LA3014 (29) Hsp7Q »ubiquitin ubiquitin 0bp -i-Obp nsaper R-svj10 DV316 < 3 (30) Hsp7Q- 'ubiquitin ubiquitin + 0bp ÷ 0bp rea} ) crKR-5v40 LA30S > 7 (27) Hsp70 íTAV-KlO * 0bp ÷ 0bp T..A3077 (26) Hsp70-íTAV ITAV- 10 + 0bp ÷ Qbp LA3233 (28) Hsp70 ITAV2- 10 + 0bp + 0bp LA3376 (31) Bsp70 tTAV2-K10 • i-0bp + p LA3376 (31) Sry * a t'TÁV3-sv40 + 0bp 40¾p LA3376 (3J) MB reaperK -sv4C + 0bp + 0bp LA3242 (32) HB reaperK -sv40 + 0bp ÷ 0¾p LA1038 (14) Hsp70-tra Tra-Nippl + 22bp ÷ 20bp (nipper) -sv40 LA3054 (61) 0pie2-R.ls QsRcd- tra- ubiquitin + 22bp + 20bp tra VTAY-SV40 LA3056 (62) Opie2- ls-D5 cd- trans- ubiquitin +?.?. Bp + 242bp tra tTAV-svlO LA3488 (63) Tel-nls- ubiquitin n! S- + 0bp K) bp TurboGieen-nls- DsRcd- Eis-sv40 ubiquituia LA3596 (67) lel-nls- ubiquitinanls- -Ob -t-Obp TurboGreen-nls- DsRcd-als-sv- 0 ubiquitin Table 5 As mentioned above when an itron is placed 5 'to a region of the protein coding (ORF-X), it is preferred to place or use ubiquitin 3' to the intron, 5 'to ORP-X, as well and providing the female specific regulation of ORF-X, while introducing the physical separation between that sequence and the intron tra, thereby reducing the occasions in which sequences within ORF-X will interfere with the intron splice tra. Constructs and sequences of the compound are also considered, for example of the form: X-ubi-Y with the alternatively spliced intron inserted between the coding region X and the ubiquitin coding region (ubi), or within of the coding region of ubiquitin, or between ubiquitin and the coding region Y. Thus X will be expressed independently of the splice of the intron, while Y will be expressed only when the intron is spliced in a convenient manner. Other configurations and arrangements of this general type will be apparent to the person skilled in the art. Some Examples of this are LA3014, LA3054, LA3056, LA3166, LA3488 and LA3596, all of which use the ubiquitin fusions, thus demonstrating the ability of this idea to be applied successfully in the transgenic Mediterranean fly. Alternative examples in transgenic mosquitoes include LA3604 and LA3612, demonstrating the broad phylogenetic applicability of this system in not only diverse species (mosquitoes and Mediterranean flies), but also in various contexts including AaActin4, Aadsx and Cetra. LA3596 (see Figure 67 and SEQ ID No. 145) has a design similar to LA3488, aiming to generate green fluorescence (by the expression of the fluorescent protein localized in the TurboGreen nucleus) in both sexes, but red fluorescence only in females (by the expression of the fluorescent protein located in the DsRed2 nucleus). This is achieved by the fusion of these two proteins, promoted by the Hr5-le1 enhancer / promoter cartridge, linked together with a short 11 amino acid linker (linker SG4) and a coding region encompassing ubiquitin (with a mutation point intended to stabilize the resulting protein reducing its propensity for degradation mediated with ubiquitone) and the Cetra intron to limit the expression of DsRed2 to the females. The transgenic Mediterranean fly was generated with this construction. The red fluorescence was limited to the females in this line as expected, while the Green fluorescence was observed in all males and females. This could be used for the separation by sexes by fluorescence discrimination of a particular fluorescent protein, in this case red fluorescence that represented the expression of DsRed2. Example 17: Another Cetra exemplification LA3014 and LA3166 are also referenced and the phenotypic data of these in other examples We have previously done, and we have obtained transgenic with the Cetra intron in a functional protein with the exception of tTAV, such as LA3014 and LA3166. LA3014 contains a ubiquitin-reaperK fusion downstream of a Cetra intron. The phenotypic data that the fly of the Mediterranean transgenic LA3014 produced a specific lethality to the females repressible. The RT-PCR analysis in the RNA extracted from adult males and from females reared without tetracycline, using the primers and ReaperKR, demonstrates that the correct splicing occurred in the females (508bp band) and no such band was found in the males (figure 37). LA3166 is another construct with the Cetra intron placed within the coding region of ubiquitin fused to reaperKR, but placed in a different position in ubiquitin. LA3166 also produces a lethal effect specific to the repressible females dominant in the Mediterranean fly. LAI 038 is a new example of the use of the Cetra intron in a diverse sequence context, here placed in a fragment of NippIDm called "nipper" that is also correctly spliced in the transgenic Mediterranean fly when it is analyzed by means of RT-PC (figure 12). LA670 was required as a source of tTAV to drive the expression of the nipper alternatively spliced. We have also done again, and we have obtained transgenic with, "introns only" constructs based on Cetra with the intron in a different gene (many of the previous examples, unless otherwise evident, are in the tTAV or in one of its variants, ie tTAV2 or tTAV3). These constructions work as indicated. This is an important result, thus demonstrating that there are no essential Cetra exonic sequences that we have simply duplicated (in function, if not necessarily in sequence) by chance, in tTAV. We also have ubi-rprKR constructs of this type (LA3014 and LA3166), which also validates the ubiquitin fusion method described above. The ubiquitin fusion method is further exemplified by the RT-PCR analysis of LA3054, LA3056 and LA3488 (figures 11, 13, 14), as described in example 16, above. Figure 11: Gel showing the sex-specific splice of introns derived from Cetra (780bp band in females) in Ceratitis capitata transformed with LA3488. The splice in the F1 form would produce a product of approximately 780bp. A band of this size is clearly visible from the females (lane 4), but not in the males, nor in the lanes with the reactions of which the enzyme reverse transcriptase was omitted ("without RT"). Therefore, the intron derived from Cetra is capable of the alternative splice specific to sex in this new sequence context. Lane 1: Marker (Eurogentec's SmartLadder ™, bands of approximately 0.8, 1.0 and 1.5kb are indicated); Lanes 2 and 3: Ceratitis capitata LA3488 / + males (RT control and without RT control, respectively); Lanes 4 and 5: Ceratitis capitata LA3488 / + females (control RT and without RT control, respectively). Figure 12: Gel showing the specific splice to sex of introns derived from Cetra in Ceratitis capitata transformed with LA1038. The splice in the F1 form will produce a product of approximately 230bp. A band of this size is clearly visible in females (lanes 1, 2, 7, 8, 9 and 10), but not in males. Therefore, the intron derived from Cetra is capable of the alternative splice specific to sex in this new context of the sequence. Lane 15: Marker (Eurogentec's SmartLadder ™, bands of approximately 0.2, 0.4 and 0.6kb are indicated); Lanes 1, 2, 7, 8, 9 and 10: Ceratitis capitata LA670; Females LA1038; Lanes 3, 4, 5, 6, 11, 12, 13 and 14: Ceratitis capitata LA670; LAI 038 males. Figure 13: Gel showing the sex-specific splice of introns derived from Cetra in Ceratitis capitata transformed with LA3054. The splice in the F1 form will produce a product of approximately 340bp. A band of this size is clearly visible in lane 7, but not in males. Therefore, the intron derived from Cetra is capable of the alternative splice specific to sex in this new context of the sequence. Lane 1: Marker (SmartLadder ™ of Eurogentec, bands of approximately 0.4, 0.6, 0.8 and 1.0kb are indicated); Lanes 2-5: Ceratitis capitata LA3054 males; Lane 7: Ceratitis capitata LA3054 females. Figure 14: Gel showing the sex-specific splice of introns derived from Cetra in Ceratitis capitata transformed with LA3056. The splice in the F1 form will produce a product of approximately 200 bp. A band of this size is clearly visible from a female (lane 6), but not from males (lanes 2-4). Therefore, the intron derived from Cetra is capable of the alternative splice specific to sex in this new context of the sequence. Lane 1: Marker (Eurogentec's SmartLadder ™, bands of approximately 0.2, 0.4, 0.6 and 0.8kb are indicated); Lanes 2-5: Ceratitis capitata LA3056 / + males; Lanes 6-7: Ceratitis capitata LA3056 / + females. Figure 15: Gel showing the sex-specific splice of introns derived from BzTra in Anastrepha ludens transformed with LA3376. The splice in the F1 form will produce a product of approximately 672 bp. A band of this size is clearly visible from the females (lane 4), but not in the males, nor in the lanes with the reactions of which the enzyme reverse transcriptase was omitted ("RT"), the primers used were SRY and AV3F. Therefore, the intron derived from Bztra is capable of the alternative splice specific to sex in this new context of the sequence. Lane 1: Marker (Eurogentec SmartLadder ™, bands of approximately 0.6, 0.8, and 1.0kb are indicated) Lanes 2 and 3: Anastrepha ludens LA3376 / + males (with RT control and without RT control, respectively); Lanes 4 and 5: Anastrepha ludens LA3376 / + females (with RT control and without RT control, respectively). Figure 18 and SEQ ID no. 149 and 150 show the DSX and minigen2 DSX minigene sequences and map of plasmid LA3619. Figures 19-51 correspond to examples 1-9 above. Figures 52-58, 68 and 69 show various diagrams and sequences of the plasmid. Figures 59-60 are described above and Figures 61-66 show several additional diagrams and sequences of the plasmid. Figure 67 is pLA3596, as deduced elsewhere. References Alien ML, Christensen BM. Related 2004 Flight muscle-specific expression of act88F: GFP in transgenic Culex quinquefasciatus Say (Diptera: Culicidae). Parasitol Int. 55 (4): 307-14. Bennett D5 Szoor B, Gross S, VereshchaginaN, Alphey L. 2003 Ectopic expression of inhibitors of protein phosphatase type 1 (PPI) can be used to analyze roles of PPI in Drosophila development. Genetics 164 (1): 235-45. Black, D. (2003). Mechanisms of alternative pre-messenger RNA splicing Annu Rev Biochem 72, 291-336. Burset, M., Seledtsov, L, and Solovyev, V. (2001). SpliceDB: database of canonical and non-canonical splice sites in mammalian genomes. Nucleic Acids Research 2P5 255-259. Caceres JF, Kornblihtt AR. 2002 Alternative splicing: multiple control mechanisms and involvement in human disease. Trends Gent. 18 (4): 186-93. Cande C5 Cecconi F, Dessen P5 Kroemer G. 2002 Apoptosis-inducing factor (AIF): key to the conserved caspase-independent pathways of cell death? J Cell Sci. 115 (24): 4727-34. 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DNA Res. 7: 165-174. Olson, M.R., Holley, C.L., J¡ Yoo, S., Huh, J.R, Hay, B.A. and Kornbluth, S. (2003). Reaper is regulated by IAP-mediated Ubiquitination. J.Biol.Chem., 27Sf < 5;: 4028-4034. Olson, M.R., Holley, CX., Gan, E.C., Colon-Ramos, D.A., Kaplan, B. and Kornbluth, S. (2003). A GH3-like domain n reaper is required for mitochondrial localization and nduction of IAP degradation. J. Biol. Chem.218 (45). -44758-44768. Pan, Q., Shai, O., Misquitta, C, Zhang, W., Saltzman, A., Mohammad, N., Babak, T., Siu, H., Hughes, T., Morris, Q., et. to the. (2004). Revealing global regulatory features of mammalian alternative splicing using a quantitative microarray platform. Mol Cell 16, 929-941. Pane, A., Salvemini, M., Delli Bovi, P., Polito, C, and Saccone, G. (2002). The transformer gene in Ceratitis capitata provides a gentic basis for selecting and remembering the sexual fate. Development 129, 3715-3725. Park, J., Parisky, K., Celotto, A., Reenan, R., and Graveley, B. (2004). Identification of alternative splicing regulators by RNA interference in Drosophila. Proc Nat'l Acad Sci (USA) 101, 15974-15979. Parker L, Gross S, Beullens M, Bollen M, Bennett D, Alphey L. 2002 Functional interaction between nuclear inhibitor of protein phosphatase type 1 (NIPPI) and protein phosphatase type 1 (PPI) in Drosophila: consequences of over-expression of NIPPI in flies and suppression by co-expression of PPI. Biochem J. 368 (3): 1 '89 -97.

Raphael, K.A., Whyard, S., Shearman, D., An, X. and Frommer, M. (2004). Bactrocera tyroni and closely related pest-tephritids-molecular analysis and prospects for transgenic control strategies. Insect Biochem. Mol. Biol. 34: 167-176. Ryner, L. and Baker, B. S. (1991). Regulation of doublesex pre-mRNA processing occurs by 3'- splice site activation. Genes Dev. 5: 2071-2085. Saccone, G., Pane, A., and Polito, C. (2002). Sex determination in flies, fruitfles and butterflies. Genetics 116, 15-23. Scali, C, Catteruccia, F., Li, Q., and Crisanti, A. (2005). Identification of sex-specific transcripts of the Anopheles gambiae doublesex gen. J Exp Biol 208, 3701-3709. Scott, M., Heinrich, J., and Li, X. (2004). Progress towards the development of a transgenic strain of the Australian sheep blowfly (Lucilia cupnna) suitable for a male-only sterile relay program. Insect Biochem Mol Biol 34, 185-192. Seo, S-J., Cheon, H-M., Sun, J., Sappington, T. W. and Raikhel, A.S. (2003). 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The STAR / GSG family protein rSLM-2 regulates the selection of alternative splice sites. J. Biol. Chem. 276 (12): 8665-8673. Streuli, M. and Saito, H. (1989). Regulation of tissue-specific alternative splicing: exon-specific cis-elements govern the splicing of leukocyte common antigen pre-mRNA. EMBO J. 8 (3): 787-796. Suzuki, M., Ohbayashi, F., Mita, K., and Shimada, T. (2001). The mechanism of sex-specific splicing at the doublesex gen is different between Drosophila melanogaster and Bombyx mori. Insect Biochem Mol Biol 31, 1201-1211. Thanaraj, T., and Clark, F. (2001). Human GC-AG alternative intron isoforms with weak donor sites show enhanced consensus at acceptor exon positions. Nucleic Acids Research 29, 2581-2593. Thanaraj, T., Stamm, S., Clark, F., Reithoven, J., Le Texier, V., and Muilu, J. (2004). ASD: the Alternative Splicing Datábase. Nucleic Acids Research 32, D64-D69. Varshavsky, A. (2000). Ubiquitin fusion technique and its descendants. Meth Enz 327. Venables, J. (2002). Alternative splicing in the testes. Curr Opin Gent 12 Dev, 615-619. Venables JP. 2004 Aberrant and alternative splicing in cancer. Cancer Res.64 (21): 7647-54. Vernooy, S.Y., Copeland, J., Ghaboosi, N., Griffin, E.E., Yoo, SJ. and Hay, BA. (2000). J. Cell Biol. 150 (2): F69- ¥ 75. White, K., Tahoaglu, E. and Steller, H. (1996). Cell killing by the Drosophila gen reaper. Science 271 (5250): 805-807. Wing, J.P., Zhou, L., Schwartz, L.M. and Nambu, J.R. (2001) Distinct cell killing properties of the Drosophila reaper, head involution defective, and grim genes. Cell Death Diffn 5 (11): 930-939 Yali Chiu A., and Pin Ouyang, A. B., (2006). Loss of Pnn expression attenuates expression levéis of SR family splicing factors and modulates alternative pre-mRNA splicing in vivo. Bioch. Biophys.Res. Comm.iW: 663-67. Yoshimura, K., Yabuta, Y., Ishikawa, T. and Shigeoka, S. (2002). Idenitization of a cis element for tissue-specific alternative splicing of chloroplast Ascorbate Peroxidase pre-mENE in higher plants. J. Biol. Chem 277 (43), 1: 40623-40632. SEQUENCE ANNOTATIONS The following refers to the various plasmids herein and highlights the position of certain preferred elements herein. <; 223 > Sequence of pLA3359 (SEQ ID NO.47). < *** > Key features include: 1. Mini-gene dsx from Anopheles gambiae (Agdsx), [a mini-gene is a recombinant sequence derived from a particular gene (the Agdsx gene in this example) linking together non-contiguous segments while retaining the original order 5'- 3 'order; this is equivalent to the deletion of some internal segments of a longer fragment of the genomic sequence derived from the gene], (1-3135): including the Agdsx part of exon3, of exon 4a (female), of exon 4b (female) and from the part of exon5 (male and female). < *** > Exons derived from Agdsx from positions 426 to 560 (part of exon 3); 1068 to 2755 (including part of exon 4, found in females); 1809 to 2755 (including part of exon 4, found in females); and 2914 to 3135 (including part of exon 5, found in males). < *** > The alternatively spliced transcription starts in the segment derived from the baculovirus AcMNPV read (immediately before 1) at position -8031 (the fragment of lei is from position 7431 to 8060). < *** > Included caraceristics: 1. The additional intron derived from the Drosophila scraps ('intron scraps') gene upstream to the Agdsx sequence from position 8075 to 8137. < 223 > Sequence of pLA3433 (SEQ ID NO 48). < *** Key features include: 1. Agdsx Mini-gene (778-4623): including the Agdsx part of exon 2, exon3, exon 4a (female), exon 4b (female) and part of exon5 (male and female) ). < *** > Exons derived from Agdsx from position 778 to 908 (part of exon 2); 1913 to 2048 (part of exon 3); 2556 to 2642 (part of exon 4a); 3297 to 4243 (part of exon 4b) and 4402 to 4623 (part of exon 5). < *** > The alternatively spliced transcription starts in the baculovirus derived segment AcMNPV read (immediately before 1) at position -606 (the lei fragment is position 6 to 635). < *** > Feature included: 1. The additional intron derived from the Drosophila scraps ('intron scraps') gene upstream to the Agdsx sequence from position 650 to 712. < 223 > Sequence of pLA3491. < *** > Key features include: 1. Aedes aegypti dsx mini-gene (Aadsx): including part of exon 4, of exon5a (female), of exon 5b (female), and part of exon 6 (male and female) of Aadsx. < *** > Exons derived from Aadsx from position 1316 to 1450 (part of exon 4); 2626 to 3761 (part of exon 5a); 3293 to 3761 (part of exon 5b); and 5215 to 5704 (part of exon 6). < *** > The part of the F1 transcript is predicted to comprise the nucleotides -1174-1450, 2626-3761, 5215-5850. < *** > The part of the F2 transcript is predicted to comprise the nucleotides -1174-1450, 3293-3761, 5215-5850. < *** > The part of the F3 transcript is predicted to comprise the nucleotides -1174-1450, 2626-3083, 3293-3761, 5215-5850. < *** > The part of the M1 transcript is predicted to comprise the nucleotides -1174-1450, 5215-5850. < *** > The alternatively spliced transcription starts in the baculovirus derived segment AcMNPV read (immediately preceding 1) at position -1174 (the lei fragment is position 574 to 1203). < *** > Feature included: 1. The additional intron derived from the Drosophila scraps ('intron scraps') gene upstream to the Agdsx sequence from position 1218 to 1280. < 223 > Sequence of pLA3646. < *** > Key features include: 1. Aadsx Mini-gene (17218-11707): including part of position 17113 to 16979 of exon 4, exon 5a of position 15803 to 15025 to + 14010 to 13650, exon 5b of position 15136 to 15025 to + 14010 a to 13650 and to position 12196 to 11707 of exon 6 (note: reverse orientation) of Aadsx. < *** > the exon 4 part contains 4 point mutations relative to the wild type at positions 17087 (ATG-ACG), 17053 (ATG-ACG), 17050 (ATG-ACG) and 17041 (ATG-ACG) (note: reverse orientation); the part of exon 5a and 5b contains 3 stern mutations in relation to the wild type at positions 15129 (ATG-ATA), 15116 (ATG-ATA) and 15113 (ATG-ATA) (note: reverse orientation). All these mutations are to eliminate the ATG sequences. tTAV2 is inserted into overlapping exons 5a and 5b from position 15024 to 14011 (note: reverse orientation). < *** > The alternatively spliced transcript starts at the fragment derived from hsp70 at position ~ 17312 (the hsp70 fragment is from position 17354 to 17225); (note: reverse orientation). < *** > Feature included: 1. The additional intron derived from the Drosophila scraps ('intron scraps') gene upstream to the Agdsx sequence from position 1107 to 1045. (note: reverse orientation). Sequence of pLA3435 (SEQ ID NO.46) < *** > Key features include: 1. Bombyx mori dsx (Bmdsx) mini-gen (1411-3161): with an exogenous link between the female exons 3 and 4 &4. < *** > Fragment of shared exon two (1411 bp-1554bp) < *** > The part of the female specific exon three (2121 bp-2202) fuses the specific female exon 4 (2225bp-2290bp) using an exogenous binder (2203bp-2224bp) <; *** > fragment of shared exon five (3007bp-3161 bp) < *** > | jn splicing product of the female dsx mini-gene is coded by 1411-1554 + 2121-2290 + 3007- 3161. < *** > A splicing product of the male dsx mini-gene is encoded by 1411-1554 + 3007-3161. < *** > Transcription is predicted to start at approximately position -1239 within the segment derived from the baculovirus promoter AcMNPV read (immediately preceding 1) (639bp-1268bp). < 223 > Sequence of pLA3534. < *** > Key features include: 1. Aadsx Mini-gene (6996-4425): containing exon 4, the exon5a part (female) and the exon 5b part (female), of Aadsx that including fragments of the Aadsx intron. < *** > Exons derived from Aadsx from position 6968 to 6834 (part of exon 4), 5462 to 4425 (part of exon 5a) and 4795 to 4425 (part of exon 5b); (note: reverse orientation). < *** > Part of the F1 transcript is predicted to comprise nucleotides -7146-6834, 5462-4300 (note: reverse orientation). < *** > Part of the F2 transcript is predicted to comprise nucleotides -7146-6834, 4795-4300 (note: reverse orientation). < *** > Part of the F3 transcript is predicted to comprise the nucleotides -7146-6834, 5462-5005, 4795-4300 (note: reverse orientation). < *** > The alternatively spliced transcription starts in the segment derived from the baculovirus AcMNPV read (immediately preceding 1) at position 7146 (the fragment of lei is from position 7746 to 7117, reverse orientation). < 223 > Sequence of pLA3612. < *** > Key features include: 1. Ubiquitin-tTAV2 coding region inserted into a female exon of the Aadsx gene. < *** > Ubiquitin-tTAV2 is found from position 15185-16429 in Aadsx (ubiquitin is from 15185- 15412; tTAV2 is found in 15413-16429), including start and stop codons. < *** > Sequence derived from Aadsx: 13150-15184, 16438-18805. < *** > The alternatively spliced Aadsx-ubiquitin-tTAV2 transcript starts in the hsp70-derived segment (the hsp7Q fragment is found at 13014-13143). < 223 > Sequence of pLA3619. < *** > Key features include: 1. tTAV2 coding region inserted into a female exon of the Aadsx gene. < *** > Sequence derived from Aadsx: 5635-364I5 2610-243 (note: reverse orientation). < *** > The alternately spliced Aadsx-tTAV2 transcript starts at the hsp70 derived segment from 5642-5771 (note: reverse orientation). < *** > It is predicted that the tTAV2 transcript is translated between 2619-3635, including the start and stop codon (note: reverse orientation). < 223 > Sequence of pLA3545. < *** > Key features include: 1. Promoter AaActin4 and 5 'UTR including the first intron regulates the expression of tTAV. < *** > The sequence derived from AaActin4 is found at position 923-4285. < *** > The alternatively spliced transcript is predicted to start at approximately -2366. < *** > £ | The first intron of AaActin4 (female variant of the splice) extends from 2458-4259. < *** > It is predicted that tTAV moves between 4300-5316, including the start and stop codon. < 223 > Sequence of pLA3604. < *** > Key features include: 1. Promoter AaActin4 and 5 'UTR regulates the expression of ubiquitin-tTAV2. < *** > The sequence derived from AaActin4 extends position 5795-2407 (note: reverse orientation). < *** > It is predicted that the alternatively spliced transcript starts at approximately ~ 4353 (note: reverse orientation). < *** > The first intron of AaActin4 (female variant of the splice) extends from 2455-4254 (note: reverse orientation). < *** > Ubiquitin-tTAV2 transcription is predicted to move from a directed start codon in the first exon of the AaAct4 gene at 4299-4297 (the ubiquitin extends 2406-2179, tTAV2 extends from 2178-1162); (note: reverse orientation). < 223 > Sequence of pLA364. < *** > Key features include: 1. Region of tTAV coding inserted into a female exon of the CodlingDsx gene. < *** > tTAV extends position 2731-3747 in the CodlingDsx gene. < *** > Transcript Dsx-tTAV alternatively spliced starts in the derived segment of hsp70 (the hsp70 fragment extends from 4811-4940). < *** > The transcription of tTAV is predicted to be translated between 2731-3747, including the start and stop codon (note: reverse orientation). < 223 > Sequence of pLA3570. < *** > Key features include: 1. Region of tTAV coding inserted into a female exon of the PBX-Dsx gene. < *** > tTAV extends position 2336-3352 < *** > The transcript Dsx-tTAV alternatively spliced start at the derived segment hsp70 (fragment hsp70 extends 4683-4812). < *** > Transcription of tTAV is predicted to be transferred between 2336-3352, including the start and stop codon (note: reverse orientation). < 223 > Sequence of pLA1118 (SEQ ID NO.49). < *** > Key features include: 1. tTAV coding region with the intron inserted from Cetra. < *** > The intron of Cetra is from position 3905-2561 in the tTAV (note: reverse orientation). < *** > Transcription of the alternatively spliced tTAV starts at the hsp70 derived segment at position 4217 (the hsp70 fragment extends from 4260-4131); (note: reverse orientation). < *** > The tTAV F1 transcript is predicted to be moved between 4040 - 1679 (note: reverse orientation). < *** > Feature included: 1. Intron Adh within the expected F1 transcript of position 4118-4049 (note: reverse orientation). < 223 > Sequence of pLA3077 (SEQ ID NO.50). < *** > Key features include: 1. tTAV coding region with the intron inserted from Cetra. < *** > The intron of Cetra is from position 3975-2631 in the tTAV (note: reverse orientation). < *** > The transcript of the alternatively spliced tTAV starts in the derived segment hsp70 at position ~ 4217 (the hsp70 fragment extends from 4260-4131); (note: reverse orientation). < *** > The tTAV F1 transcript is predicted to be translated between 4039 - 1678, including the start and stop codon (note: reverse orientation). < *** > Feature included: 1. Adh intron within the expected F1 transcript of position 4117-4048 (note: reverse orientation). < 223 > Sequence of pLA3097 (SEQ ID No. 51). < *** > Key features include: 1. tTAV coding region with the intron inserted from Cetra. < *** > The intron of Cetra is from position 3282-1938 in the tTAV (note: reverse orientation). < *** > the transcript of the alternatively spliced tTAV is predicted to start in the derived segment hsp70 at position -3382 (the hsp70 fragment extends from 3425-3296); (note: reverse orientation). < *** > tTAV F1 will be moved between 3285 - 924, including the start and stop codon (note: reverse orientation). < 223 > Sequence of pLA3097 (SEQ ID NO.52). < *** > Key features include: 1. tTAV2 coding region with the intron inserted from Cetra. < *** > The Cetra intron is from position 3289-1945 in tTAV2 (note: reverse orientation). < *** > Transcription of alternatively spliced tTAV2 starts at the derivative segment hsp70 at position -3389 (the hsp70 fragment extends from 3432-3303); (note: reverse orientation). < *** > The tTAV2 F1 transcript is predicted to be translated between 3285-931, including the start and stop codon (note: reverse orientation). < 223 > Sequence of pLA3014 (SEQ ID NO 53). < *** > Key features include: 1. Ubi-reaper coding region [KR] with the Cetra intron inserted. < *** > The intron Cetra is at position 3356-4700 in the ubi-reaper [KR]. < *** > transcription of ubi-reaper [KR] alternatively spliced starts in the segment derived from hsp70 in the position ~ 3234 (the hsp70 fragment is found from 3191-3320). < *** > it is predicted that the transcription of the ubi-reaper [KR] F1 will be transferred between 3331-5143, including the start and stop codon (ubiquitin is found from 3331-3355, 4701-4948, the reaper [KR] it is found from 4949 - 5143). < 223 > Sequence of pLA3166 (SEQ ID No. 54). < *** > Key features include: 1. Ubi-reaper coding region [KR] with the Cetra intron inserted. < *** > The Cetra intron is in position 9987-8643 in the location reaper [KR]. (note: reverse orientation). < *** > transcription of alternatively spliced ubirer [KR] starts in the segment derived from hsp70 at position ~ 10227 (the hsp70 fragment is found from 10270-10141). (note: reverse orientation). < *** > it is predicted that transcription of the ubi-reaper [KR] F1 will be transferred between 10126-8359, including the start and stop codon (ubiquitin is found from 10126-9988, 8642-8554, the reaper [KR] it is found from 8553 - 8359). (note: reverse orientation). < 223 > Sequence of pLA3376 (SEQ ID No. 55). < *** > Key features include: 1. tTAV2 coding region with the Cetra intron inserted. 2. tTAV3 coding region with the Bztra intron inserted. 3. region of the coding reaper [KR] with the intron Bztra inserted. < *** > The intron of Cetra is from position 3289-1945 in tTAV2 (note: reverse orientation). < *** > The Bztra intron is found from position 5981-5014 in tTAV3 (note: reverse orientation). < *** > The Bztra intron is found from position 16391-17358 in the reaper [KR]. < *** > transcription of alternatively spliced tTAV2 starts in the segment derived from hsp70 at position ~ 3389 (the hsp70 fragment is found from 3432-3303). (note: reverse orientation). < *** > transcription of alternatively spliced tTAV3 starts in the segment derived from sry-alpha at position ~ 3389 (the sry-alpha fragment is found from 6243-5999). (note: reverse orientation). < *** > the alternately spliced reaper [KR] transcript starts in the hunchback-derived segment at position ~ 16339 (the hunchback fragment is found from 16289-16372). (note: reverse orientation). < *** > it is predicted that the transcription of tTAV2 F1 will be transferred between 3292-931, including the start and stop codon (note: reverse orientation). < *** > it is predicted that the transcription of tTAV3 F1 will be transferred between 5984-4006, including the start and stop codon (note: reverse orientation). < *** > it is predicted that the transcription of reaper [KR] F1 will be transferred between 16385-17550, including the start and stop codon (note: reverse orientation). < 223 > Sequence of pLA3342 (SEQ ID NO 56). < *** > [The key features include: 1. tTAV coding region with the Cetra intron inserted. 2. region of the reaper coding [KR] with the intron Cetra inserted. < *** > The intron of Cetra is from position 3282-1938 in tTAV (note: reverse orientation). < *** > The neutron of Cetra is located from position 5488-4180 in the reaper [KR]. (note: reverse orientation). < *** > the alternately spliced reaper [KR] transcript starts at the hunchback-derived segment at the ~ 5540 position (the hunchback fragment is found from 5590-5507). (note: reverse orientation). < *** > transcription of alternatively spliced tTAV starts in the segment derived from hsp70 at position ~ 3382 (the hsp70 fragment is found from 3425-3296). (note: reverse orientation). < *** > it is predicted that the transcription of reaper [KR] F1 will be transferred mainly between 4088-5494, including the start and stop codon (note: reverse orientation). < *** > it is predicted that the transcription of tTAV F1 will be transferred mainly between 924-3285, including the start and stop codon (note: reverse orientation). < 223 > Sequence of pLA1172 (SEQ ID NO 106). < *** > Key features include: 1. tTAV coding region between fragments derived from AaActin4. < *** > Fragments derived from AaActin4 are counted from 7868-11257 and 12366-13100. < *** > it is predicted that the transcription of tTAV will be transferred mainly between 11342-12358, including the start and stop codon < *** > it is predicted that the transcription of AaActin4 starts at position ~ 9312. < *** > AaActin4 contains an intron (female type splice variant) from position 9403-11204. < 223 > Sequence of pLA1038 (SEQ ID NO.12). < *** > Key features include: 1. coding region of the NippIDm fragment ("nipper") with the Cetra intron inserted with the flanking tra / exonic sequence; *** > The intron of Cetra is found from position 3365-4709 in nipper. < *** > The Ccctra intron is flanked by the Cetra exonic sequence at positions 3343-3364 and 4710-4729. < *** > the transcript of alternatively spliced nipper starts in the segment derived from hsp70 at position ~ 3243 (the hsp70 fragment is found from 3200-3329). < *** > it is predicted that the transcription of nipper F1 will be transferred mainly between 3340-5014, including the start and stop codon. < 223 > Sequence of pLA3054 (SEQ ID NO 158). < *** > The key features include: 1. coding region of the DsRed-ubi-tTAV fragment with the Cetra intron inserted with the exonic flanking tra sequence < *** > The intron of Cetra is found from position 3509-2165 in DsRed-ubi-tTAV. < *** > The Ccctra intron is flanked by the exonic sequence Cetra at positions 3531-3510 and 2164-2145. (note: reverse orientation). < *** > transcription of alternatively spliced DsRed-ubi-tTAV starts either in the derivative segment of hsp70 at position ~ 3245 (the hsp70 fragment is found from 4930-4801) or in the Opie2-derived segment at position ~ 4353 ( the Opie2 fragment is found from 4795-4255) (note: reverse orientation). < *** > it is predicted that the transcription of DsRed-ubi-tTAV F1 will be mainly transferred between 4320-888, including the start and stop codon (DsRed is 4212-3538, ubiquitin is from 2135-1908, tTAV is found in 1907-888). (note: reverse orientation). < 223 > Sequence of pLA3056 (SEO, ID NO 159). < *** > The key features include: 1. Coding region of the DsRed-ubi-tTAV fragment with the Cetra intron inserted with the exonic flanking tra sequence < *** > The intron of Cetra is found from position 3731-2387 in DsRed-ubi-tTAV. (note: reverse orientation). < *** > f £ | Ccctra is flanked by the exonic sequence Cetra at positions 3753-3732 and 2386-2145. (note: reverse orientation). < *** > transcription of alternatively spliced DsRed-ubi-tTAV starts either in the derived segment of hsp70 at the > ~ 5109 (the hsp70 fragment is found from 5152- 5023) or in the Opie2 derived segment at position ~ 4575 (the Opie2 fragment is found from 5017-4477) (note: reverse orientation). < *** > it is predicted that the transcription of DsRed-ubi-tTAV F1 will be transferred mainly between 4542-888, including the start and stop codon (DsRed is 4434-3760, ubiquitin is from 2135-1908, tTAV is found in 1907-888). (note: reverse orientation). < *** > Feature included: 1. additional intron derived from the Cetra gene (second intron of the Cetra F1 transcript) within the F1 transcript predicted from position 2222-2168 (note: reverse orientation). < 223 > Sequence of pLA3488 (SEO, ID NO 160). < *** > Key features include: 1. Coding region of. TurboGreen-ubi-DsRed with the intron Cetra inserted. < *** > The Cetra intron is found from position 2263-3607 in TurboGreen-ubi-DsRed. < *** > The alternatively spliced transcription of TurboGreen-ubi-DsRed begins in the baculovirus derived segment AcMNPV read (immediately preceding 1) at position ~ 1180 (the fragment of lei is from position 580 to 1209). < *** > It is noted that the TurboGreen-ubi-DsRed F1 transcript will be translated between 1311-4467, including the start and stop codon (TurboGreen is from 1311-2093; linker SG4 it is from 2094-2123; Ubiquitin is found from 2124-3696, including Cetra intron; DsRed is from 3697-44-67). < *** > Included feature: 1. Additional intron derived from the Drosophila scraps gene ("intron serps") within the F1 transcript predicted from position 1224-1286. < 223 > Sequence of pLA3596 (SEQ ID NO.145). < *** > Key features include: 1. TurboGreen-ubi-DsRed2 coding region with the Cetra intron inserted. < *** > The Cetra intron is found from position 5947-7291 in TurboGreen-ubi-DsRed2. < *** > The alternately spliced transcript of TurboGreen-ubi-DsRed2 begins in the baculovirus derived segment AcMNPV read (immediately preceding 1) at position -4864 (the lei fragment is from position 4264-4893). < *** > It is noted that the TurboGreen-ubi-DsRed2 F1 transcript will be translocated between 4995-8148, including the start and stop codon (TurboGreen is from 4995-5777, linker SG4 is found from 5778-5807, ubiquitin is found from 5808-7380 , including Cetra intron; DsRed2 is found from 7381-8151). < *** > Feature included: 1. Additional introns derived from the Drosophila scraps gene ("intron") scrps ") within the F1 transcript predicted from position 4908-4970 2. Assumed amino acid mutation compared to LA3488 at position 7294-7296.

Claims

CLAIMS 1. A polynucleotide expression system comprising: at least one heterologous polynucleotide sequence encoding a functional protein, defined between a start codon and stop codon, and / or polynucleotides for interfering RNA (RNAi), that is going to be expressed in an organism; at least one promoter operatively linked; and at least one splice control sequence that in cooperation with a spliceosome, is capable of (i) mediating the splicing of an RNA transcript of the coding sequence to yield a first spliced messenger RNA product (mRNA) and (ii) mediating at least one alternative splicing of the RNA transcript to produce an alternative spliced mRNA product; wherein when at least one heterologous polynucleotide sequence encodes a functional protein, at least one of the mature mRNA products comprises an open reading frame (ORF) that extends from the start codon to the stop codon, defining a protein which is the functional protein, or is related to the functional protein by means of at least one amino acid deletion and which is functional when transferred and optionally has undergone a post-translational modification; the measurement is selected from the group consisting of: sex-specific mediation, stage-specific mediation, mediation specific to the germ line, mediation specific to the tissue, and their combinations
2. A polynucleotide expression system according to claim 1, wherein the measurement is sex specific.
3. A polynucleotide expression system according to claim 1 or 2, wherein the polynucleotide sequence to be expressed comprises two or more coding exons for the functional protein.
4. A polynucleotide expression system according to any of the preceding claims, wherein the protein is a marker, or has a lethal, damaging or sterilizing effect.
5. A polynucleotide expression system according to claim 4, wherein the protein has a lethal effect which results in sterilization.
6. A polynucleotide expression system according to claim 5, wherein the lethal effect of the protein can be suppressed conditionally.
7. A polynucleotide expression system according to claim 4, wherein the protein is selected from the group consisting of an apoptosis induction factor, Hid, Reaper (Rpr) and NippIDm.
8. A polynucleotide expression system according to any of the preceding claims, wherein the system comprises at least one positive feedback mechanism, at least one functional protein to be differentially expressed by alternative splicing and at least one promoter thereof, wherein a product of a gene to be expressed serves as a positive transcription control factor for at least one promoter, and wherein the product or expression of the product is controllable.
9. A polynucleotide expression system according to claim 8, wherein a reinforcement is associated with the promoter, the genetic product serves to elevate the activity of the promoter by means of reinforcement.
10. A polynucleotide expression system according to claim 9, wherein the control factor is the tTA gene product or its analogue, and wherein one or more operator units teto is operably linked to the promoter and its enhancer, tTA or its analogue serve to improve promoter activity via tetO.
11. A polynucleotide expression system according to any of the preceding claims, wherein the functional protein itself is a transcriptional transactivator such as the system is tTAV consisting of tTAV, tTAV2 or tTAV3.
12. A polynucleotide expression system according to any of the preceding claims, wherein the promoter can be activated by environmental conditions, for example the presence or absence of a particular factor such as tetracycline in the tet system or by average of the variation of the environmental temperature.
13. A polynucleotide expression system according to any of claims 1-11, wherein the promoter is selected from the group consisting of the srya promoter specific to the embryo or its homologs, the Drosophila slow as molasses (slam) gene or its homologs.
14. A polynucleotide expression system according to any of the preceding claims, further comprising a boost.
15. A polynucleotide expression system according to any of the preceding claims, wherein the measurement of the alternative splice is sex specific and the splicing control sequence is derived from an intron tra.
16. A polynucleotide expression system according to claim 15, wherein the alternative splicing control is derived from the Cetra transforming gene from the Mediterranean fly, or from another ortholog or homologous Drosophila transforming gene.
17. A polynucleotide expression system according to claim 16, wherein the other ortholog or homologous of the Drosophila transforming gene is from a tephritida fruit fly.
18. A polynucleotide expression system according to claim 17, wherein the fruit fly tephritide is C, pink or B. zonata.
19. A polynucleotide expression system according to any of claims 1-14, wherein the splicing control sequence is derived from the alternative splice mechanism of the Actin-4 gene.
20. A polynucleotide expression system according to claim 19, wherein the Actin-4 gene is from Aedes spp.
21. A polynucleotide expression system according to claim 19, wherein the Actin-4 gene is from Aedes aegyti AeActin-4.
22. A polynucleotide expression system according to any of claims 1-14, wherein the splicing mechanism comprises at least one fragment of the preferred doublesex (dsx) gene which is derived from Drosophila, B. mori, worm rose, moths of apples, or a mosquito, in particular / Aedes gambiae or especially / Aedes aegypti.
23. A polynucleotide expression system according to any of claims 19-22, wherein the splice control sequence and the heterologous polynucleotide sequence encode a functional protein defined between a start codon and a stop codon, and / or polinoclecotides for RNA interference (RNA1), which are to be expressed in an organism are provided in the form of a minigene construct or a cartridge exon.
24. A polynucleotide expression system according to claim 4, wherein the system is a plasmid or a construct selected from the group consisting of any of Figures 16-18, 22-24, 26-32, 49, 52 -55, and 61-69, and / or SEQ ID NOS. 46-48, 50-56, 143-145 and 151-162.
25. A polynucleotide expression system according to any of the preceding claims, wherein the when less a splice control sequence is intronic and comprises at its 5 'end a guanine nucleotide (G), in RNA.
26. A polynucleotide expression system according to any of the preceding claims, wherein the at least one splicing control sequence is intronic and comprises at its 5 'end UG and UT nucleotides at its 3' end, in RNA .
27. A polynucleotide expression system according to any of the preceding claims, wherein the mediation is sex specific and is mediated or further controlled by binding the TRA protein or the TRA / TRA complex, or their homologs
28. A The polynucleotide expression system according to claim 27, wherein the system comprises a consensus sequence: TCWWCRATCAACA (SEQ ID No. 1), wherein W = A or T and R = A or G.
29. A system of polynucleotide expression according to any of the preceding claims, wherein the organism is a mammal, a fish, an invertebrate, an arthropod or an insect or plant.
30. A polynucleotide expression system according to any of the preceding claims, wherein the organism is an insect of the order Diptera.
31. A polynucleotide expression system according to claim 30, wherein the insect is a fruit fly of tefiritida selected from the group consisting of the Mediterranean fly (Ceratitis capitata), preferably mex fly (Anastrepha ludens), preferably oriental fruit fly (Bactrocera dorsalis), olive fly (Bactrocera oleae), melon fly (Bactrocera cucurbitae), fly of the fruit of Natal fly (Pink Ceratitis), cherry fly (Rhagoletis cerasi), fruit fly of Queensland (Bactrocera tyron'í), fruit fly of the peach (Bactrocera zonata) Caribbean fruit fly (Anastrepha suspensa ) or fruit fly of the West Indies (Anastrepha obliqua).
32. A polynucleotide expression system according to claim 30, wherein the insect is a mosquito of the genus Stegomyia, Aedes, Anopheles or Culex.
33. An expression system of polynucleotide according to claim 32, wherein the mosquito is selected from Aedes aegypti, Stegomyia albopictus, Anopheles stephensi, Anopheles albimanus and Anopheles gambiae.
34. A polynucleotide expression system according to claim 30, wherein the insect is selected from the group consisting of the new world worm (Cochliomyia hominivorax), the old world worm (Chrysomya bezziana) and the Australian fly of the wool cattle (Lucilia cuprina). Lepidoptera and coleoptera are also preferred, especially moths are especially preferred, including apple moth (Cydia pomonella), and silkworm (Bombyx mori), pink worm (Pectinophora gossypiella), the diamond moth (Plutella xylostella), the gypsy moth (Lymantria dispar), the orange worm (Amyelois transitella), peach tree borer (Anarsia lineatella) and the rice stem borer (Tryporyza incertulas ), also nocturnal moths, especially Heliothinae. Among the coleoptera, the Japanese beetle (Popilla japonica), striped beetle (Graphognatus spp.), Weevil (Anthonomous grandis), corn rootworm (Diabrotica spp) and Colorado potato worm (Leptinotarsa decemlineata).
35. A polynucleotide expression system according to claim 30, wherein the insect is not a Drosophilide.
36. A polynucleotide expression system according to any of the preceding claims, wherein the expression of a heterologous polynucleotide sequence leads to a phenotypic consequence in the organism. .
37. A polynucleotide expression system according to claim 31 or 2, wherein the polynucleotide sequence to be expressed comprises polynucleotides for interfering RNA (RNAi).
38. A method for controlling the population of an organism in a natural environment, which comprises: i) breeding a stock of the organism, the organism carrying a gene expression system that spans a system according to claims 1 to 36 which is a dominant lethal genetic system, i) distribute reserve animals in the environment in an area for population control; and iii) to achieve population control through lethality in the early stages by means of the expression of the lethal system in the descendant resulting from the crossing of the individuals of that reserve with the individuals of the opposite sex of the native population.
39. A method according to claim 38, in which lethality at early age is embryonic or before sexual maturity.
40. A method according to claim 39, in which the lethality at an early age occurs at the start of development. 41 A method according to claim 38 or 39, in which the lethal effect of the system is conditional and occurs in the natural environment through the expression of a lethal gene, the expression of the lethal gene is under control of a repressible transactivator protein, the rearing is performed under permissive conditions in the presence of a substance, the substance is absent from the natural environment and is capable of repressing the transactivator. 42. A method for biological control, comprising: i) the rearing of a stock of male and female organisms transformed with the expression system according to claims 1 to 36 under permissive conditions, allowing the survival of males and of hem bras, to provide a dual sex biological control agent; ii) optionally before the next step that imposes or allows the restrictive conditions to cause the death of individuals of one sex and providing a biological control agent of a single sex that includes individuals of the other sex carrying the conditional lethal genetic system; iii) release of the biological control agent of the dual sex or of a single sex in the environment in a specific place for biological control; and iv) achieve biological control with the expression of the genetic system in descendants resulting from the crossing of the individuals of the biological control agent with the individuals of the opposite sex of the native population. 43. A method of sex separation that includes: i) raising a stock of male and female organisms transformed with the expression system according to claims 1 to 36 under permissive or restrictive conditions, allowing the survival of males and females; and ii) eliminate the permissive or restrictive conditions to induce the lethal effect of the lethal gene in one sex and not in the other by means of the alternative connection specific to the sex of the lethal gene. 44. A method for biological or population control; i) raising a stock of male and female organisms transformed with the expression system according to claims 1 to 36 under permissive or restrictive conditions, allowing the survival of males and females; ii) eliminate the perm isive or restrictive conditions to induce the lethal effect of the lethal gene in one sex and not in the other by alternative, sex-specific splicing of the lethal gene that produces sex separation; iii) sterilize or partially sterilize the separated individuals and iv) obtain control through the release of sterile or partially sterile individuals separated within the natural environment of the organism. RES U M E N A polynucleotide expression system is provided which is capable of alternatively splicing the RNA transcripts of a polynucleotide sequence to be expressed in an organism.